Non-transformed Counterparts Research Articles

Antioxidant, enzyme inhibitory and antiproliferative activity of polyphenolic‐rich fraction of commercial dry ginger powder

SummaryPolyphenolic‐rich fraction obtained from locally produced dry ginger powder in Brahmaputra valley, India, and commercially available dry ginger (Zingiber officinale) rhizome powder consisted of [6]‐gingerol (41.9%), [6]‐shogaol (24.3%), 1‐dehydro‐6‐gingerdione (8.6%), [8]‐gingerol (7.2%), [10]‐gingerol (5.1%), [6]‐paradol (5.9%) and [4]‐gingerol (3.6%). Traces of methyl‐[6]‐gingerol and methyl‐[8]‐gingerol (both at 1.8%) were also detected. The fraction exhibited high antioxidant capacity [total phenolics (TP), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA assay)], effectively inhibited isolated digestive enzymes (α‐glucosidase, pancreatic lipase and angiotensin converting enzyme) and inhibited the proliferation of colon (HT29; IC50 of 1.06 ± 0.02 mg mL−1) and gastric (AGS IC50 of 1.29 ± 0.03 mg mL−1) adenocarcinoma cells, without affecting the proliferation of their nontransformed counterparts (IC50 > 2.0 mg mL−1). This case study demonstrates that locally produced and commercially available dry ginger powder from Brahmaputra valley, India, retains numerous food components that may enhance human health.

Hyper-dependence of breast cancer cell types on the nuclear transporter Importin β1

We previously reported that overexpression of members of the Importin (Imp) superfamily of nuclear transporters results in increased nuclear trafficking through conventional transport pathways in tumour cells. Here we show for the first time that the extent of overexpression of Impβ1 correlates with disease state in the MCF10 human breast tumour progression system. Excitingly, we find that targeting Impβ1 activity through siRNA is >30 times more efficient in decreasing the viability of malignant ductal carcinoma cells compared to isogenic non-transformed counterparts, and is highly potent and tumour selective at subnanomolar concentrations. Tumour cell selectivity of the siRNA effects was unique to Impβ1 and not other Imps, with flow cytometric analysis showing >60% increased cell death compared to controls concomitant with reduced nuclear import efficiency as indicated by confocal microscopic analysis. This hypersensitivity of malignant cell types to Impβ1 knockdown raises the exciting possibility of anti-cancer therapies targeted at Impβ1.

MEK Guards Proteome Stability and Inhibits Tumor-Suppressive Amyloidogenesis via HSF1

Signaling through RAS/MAP kinase pathway is central to biology. ERK has long been perceived as the only substrate for MEK. Here, we report that HSF1, the master regulator of the proteotoxic stress response, is a new MEK substrate. Beyond mediating cell-environment interactions, the MEK-HSF1 regulation impacts malignancy. In tumor cells, MEK blockade inactivates HSF1 and thereby provokes proteomic chaos, presented as protein destabilization, aggregation, and, strikingly, amyloidogenesis. Unlike their non-transformed counterparts, tumor cells are particularly susceptible to proteomic perturbation and amyloid induction. Amyloidogenesis is tumor suppressive, reducing in vivo melanoma growth and contributing to the potent anti-neoplastic effects of proteotoxic stressors. Our findings unveil a key biological function of the oncogenic RAS-MEK signaling in guarding proteostasis and suppressing amyloidogenesis. Thus, proteomic instability is an intrinsic feature of malignant state, and disrupting the fragile tumor proteostasis to promote amyloidogenesis may be a feasible therapeutic strategy.

Comparative incidence of cotton spider mites on transgenic Bt versus conventional cotton in relation to contents of secondary metabolites

With the wide adoption of transgenic Bacillus thuringiensis (Bt) cotton, the incidence of bollworm has reduced significantly, but secondary pests such as cotton spider mites have become serious problems in Bt cotton fields. The objective of the present study was to investigate the underlying mechanism of increased incidence of secondary pests in Bt cotton. Two transgenic cotton varieties, sGK321 and Bt-C12, and their non-transformed counterparts, SY321 and C12, were used to study differences in the incidence of spider mites in relation to secondary metabolites. Plants of each cotton cultivar were infested with five female adult spider mites and then isolated. Leaf samples with a pair of adult mites of the same age were transferred individually into Petri dishes for examination of egg laying and duration of development stages. The number of spider mites on Bt-C12 and sGK321 was more than that on C12 and SY321. The cotton spider mites feeding on Bt-C12 laid significantly more eggs than those feeding on C12; those feeding on sGK321 laid significantly more eggs than those feeding on SY321. The generation time of spider mites feeding on Bt-C12 was greatly reduced relative to those feeding on C12. Also, the generation time of mites feeding on sGK321 was shorter than those feeding on SY321. Gossypol and tannin contents in leaves of Bt-C12 were substantially lower than those in C12, and the contents in leaves of sGK321 were significantly lower than those in leaves of SY321. The occurrence of spider mites was more serious on Bt than non-Bt cotton, and the fitness of the mites on Bt cotton was higher than on non-Bt cotton. Reductions in gossypol and tannin contents in Bt cotton decreased the generation time and increased the number of eggs of cotton spider mites.

RETRACTED ARTICLE: OCT4 as a target of miR-34a stimulates p63 but inhibits p53 to promote human cell transformation

Human cell transformation is a key step for oncogenic development, which involves multiple pathways; however, the mechanism remains unclear. To test our hypothesis whether cell oncogenic transformation shares some mechanisms with the process of reprogramming non-stem cells to induced pluripotent stem cells (iPSC), we studied the relationship among the key factors for promoting or inhibiting iPSC in radiation-transformed human epithelial cell lines derived from different tissues (lung, breast and colon). We unexpectedly found that p63 and OCT4 were highly expressed (accompanied by low expressed p53 and miR-34a) in all transformed cell lines examined when compared with their non-transformed counterparts. We further elucidated the relationship of these factors: the 3p strand of miR-34a directly targeted OCT4 by binding to the 3′ untranslated region (3′-UTR) of OCT4 and, OCT4, in turn, stimulated p63 but inhibited p53 expression by binding to a specific region of the p63 or p53 promoter. Moreover, we revealed that the effects of OCT4 on promoting cell oncogenic transformation were by affecting p63 and p53. These results support that a positive loop exists in human cells: OCT4 upregulation as a consequence of inhibition of miR-34a, promotes p63 but suppresses p53 expression, which further stimulates OCT4 upregulation by downregulating miR-34a. This functional loop contributes significantly to cell transformation and, most likely, also to the iPSC process.

Normal ABL1 Is a Tumor Suppressor and Therapeutic Target In BCR-ABL1–positive Leukemias

Abstract BCR-ABL1 results from t(9;22)(q34;q11) reciprocal translocation resulting in BCR-ABL1 kinase expression, initiating chronic myeloid leukemia in chronic phase (CML-CP). At the initial stages of CML-CP both oncogenic BCR-ABL1 kinase and normal ABL1 kinase are expressed, however, loss of ABL1 kinase expression in CML-CP can result from an interstitial deletion in the normal chromosome 9 [del(9q34)] which may be combined with the transcriptional silencing of the alternative ABL1 promoter within the translocation eventually leading to disease progression and drug resistance. We found that BCR-ABL1 Abl1-/- cells generated a CML-blast phase (BP)-like disease phenotype in SCID mice compared to CML-CP-like disease from BCR-ABL1 Abl1+/+ cells. To determine the mechanisms responsible for blastic transformation of BCR-ABL1 Abl1-/- cells, we examined the role of ABL1 in proliferation, differentiation, apoptosis, genomic instability, and stemness. The presence of ABL1 inhibited proliferation in BCR-ABL1 cells as BCR-ABL1 Abl1-/- cells had higher clonogenic activity and proliferative rate compared to their wild-type counterparts. ABL1 is essential for myeloid differentiation since BCR-ABL1 Abl1-/- cells showed an immature blast phenotype when stained with Wright-Giemsa and myeloid differentiation markers Gr-1 and CD11b. ABL1 promoted apoptosis in response to genotoxic stress as revealed by reduced clonogenicity and elevated expression of p53, phosphoserine-15 p53 and activated caspase 3 in BCR-ABL1 Abl1 +/+ compared to knock-out cells. Although the absence of ABL1 did not enhance ROS and oxidative DNA damage, it appears that an impaired DNA damage response may be responsible for higher chromosome numbers and an accumulation of high numbers of chromosomal aberrations in BCR-ABL1 Abl1-/- cells. We detected an expansion of Lin-c-Kit+Sca-1+ leukemia stem cells (LSCs) in BCR-ABL1 Abl1-/- cells compared to BCR-ABL1 Abl1+/+ or non-transformed counterparts; among the LSCs, there was a higher percentage of CD34-Flt3- long-term and CD34+Flt3-short-term stem cells. These results showed that ABL1 is involved in regulating the LSC compartment in BCR-ABL1 cells. DNA microarray analysis revealed changes in mRNA levels of several genes involved in proliferation, myeloid differentiation, apoptosis, DNA damage response and stemness in BCR-ABL1 Abl1-/- cells in comparison to BCR-ABL1 Abl1+/+ cells. Together, these results demonstrated a critical role of ABL1 in BCR-ABL1-induced leukemia, prolonging survival in mice by suppressing proliferation and expansion of LSC, inducing myeloid differentiation, apoptosis and DNA damage response in BCR-ABL1 cells. Thus, it appears that ABL1 acts as a tumor suppressor in BCR-ABL1 –positive CML cells. Moreover, we hypothesized that the enhancement of the tumor suppressor function of ABL1 may have a significant impact on CML treatment. A small molecule activator of ABL1 kinase, 5-(1,3-diaryl-1H-pyrazol-4-yl)hydantoin (DPH), had been reported to interact with the myristoyl-binding site of ABL1 and destabilize the bent conformation of the α-1 helix, thereby preventing the auto-inhibitory conformation. DPH partially restored ABL1 activity in imatinib-treated cells. DPH-mediated stimulation of ABL1 tumor suppressor activity enhanced the effect of imatinib and ponatinib against CML CD34+ cells, Philadelphia chromosome-positive B-ALL (Ph+B-ALL) cells and relapsed Ph+B-ALL cells harboring T315I mutation without affecting normal counterparts. In summary, ABL1 is a potential tumor suppressor in BCR-ABL1-induced leukemia and stimulation of its function may play a significant role in the development of novel therapeutic strategies for CML and Ph+ALL. Disclosures: No relevant conflicts of interest to declare.

Generation and characterization of feline arterial and venous endothelial cell lines for the study of the vascular endothelium

BackgroundThe in vitro culture of endothelial cells (ECs) is an indispensable tool for studying the role of the endothelium in physical and pathological conditions. Primary ECs, however, have a restricted proliferative lifespan which hampers their use in long-term studies. The need for standardized experimental conditions to obtain relevant and reproducible results has increased the demand for well-characterized, continuous EC lines that retain the phenotypic and functional characteristics of their non-transformed counterparts.ResultsPrimary feline ECs from aorta and vena cava were successfully immortalized through the successive introduction of simian virus 40 large T (SV40LT) antigen and the catalytic subunit of human telomerase (hTERT). In contrast to the parental ECs, the transformed cells were able to proliferate continuously in culture. Established cell lines exhibited several inherent endothelial properties, including typical cobblestone morphology, binding of endothelial cell-specific lectins and internalization of acetylated low-density lipoprotein. In addition, the immortalization did not affect the functional phenotype as demonstrated by their capacity to rapidly form cord-like structures on matrigel and to express cell adhesion molecules following cytokine stimulation.ConclusionThe ability to immortalize feline ECs, and the fact that these cells maintain the EC phenotype will enable a greater understanding of fundamental mechanisms of EC biology and endothelial-related diseases. Furthermore, the use of cell lines is an effective implementation of the 3-R principles formulated by Russel and Burch.

Pyramiding of Bt cry1Ia8 and cry1Ba3 genes into cabbage (Brassica oleracea L. var. capitata) confers effective control against diamondback moth

A binary vector carrying Bacillus thuringiensis cry1Ia8 and cry1Ba3 genes was introduced into an inbred line of white cabbage by Agrobacterium tumefaciens-mediated transformation for control of diamondback moth (DBM, Plutella xylostella), and 14 kanamycin-resistant plantlets were obtained. Presence and expression of the cry1Ia8 and cry1Ba3 genes in five transformed plants were confirmed by polymerase chain reaction (PCR), Southern blot, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analyses. Insect bioassays showed that these transgenic plants were able to effectively control both susceptible and Cry1Ac-resistant DBM larvae as compared to non-transformed counterparts. Ten homozygous insect-resistant cabbage lines were obtained in the T2 generation through self-pollination, and molecular methods and insect bioassays. After natural infestation under greenhouse and field conditions, the pyramided lines exhibited excellent efficacy against DBM. Furthermore, data from field trials indicated that there were no significant differences in most agronomic traits between most the homozygous lines and the original variety. These transgenic lines may allow field study of resistance management strategies involving gene pyramiding and serve as novel insect-resistant resources in cabbage breeding.

Overexpression of a chitinase gene in transgenic peanut confers enhanced resistance to major soil borne and foliar fungal pathogens

A chitinase gene from rice (Rchit) was introduced into three varieties of peanut through Agrobacterium-mediated genetic transformation resulting in 30 transgenic events harboring the Rchit gene. Stable integration and expression of the transgenes were confirmed using PCR, RT-PCR and Southern blot analysis. Progeny derived from selfing of the primary transgenic events revealed a Mendelian inheritance pattern (3:1) for the transgenes. The chitinase activity in the leaves of the transgenic events was 2 to 14-fold greater than that in the non-transformed control plants. Seeds of most transgenic events showed 0–10 % A. flavus infection during in vitro seed inoculation bioassays. Transgenic peanut plants evaluated for resistance against late leaf spot (LLS) and rust using detached leaf assays showed longer incubation, latent period and lower infection frequencies when compared to their non-transformed counterparts. A significant negative correlation existed between the chitinase activity and the frequency of infection to the three tested pathogens. Three progenies from two transgenic events displayed significantly higher disease resistance for LLS, rust and A. flavus infection and are being advanced for further evaluations under confined field conditions to confirm as sources to develop peanut varieties with enhanced resistance to these fungal pathogens.

HSF1 Drives a Transcriptional Program Distinct from Heat Shock to Support Highly Malignant Human Cancers

Heat-Shock Factor 1 (HSF1), master regulator of the heat-shock response, facilitates malignant transformation, cancer cell survival, and proliferation in model systems. The common assumption is that these effects are mediated through regulation of heat-shock protein (HSP) expression. However, the transcriptional network that HSF1 coordinates directly in malignancy and its relationship to the heat-shock response have never been defined. By comparing cells with high and low malignant potential alongside their nontransformed counterparts, we identify an HSF1-regulated transcriptional program specific to highly malignant cells and distinct from heat shock. Cancer-specific genes in this program support oncogenic processes: cell-cycle regulation, signaling, metabolism, adhesion and translation. HSP genes are integral to this program, however, many are uniquely regulated in malignancy. This HSF1 cancer program is active in breast, colon and lung tumors isolated directly from human patients and is strongly associated with metastasis and death. Thus, HSF1 rewires the transcriptome in tumorigenesis, with prognostic and therapeutic implications.

Global enhancement of nuclear localization‐dependent nuclear transport in transformed cells

Fundamental to eukaryotic cell function, nucleocytoplasmic transport can be regulated at many levels, including through modulation of the importin/exportin (Imp/Exp) nuclear transport machinery itself. Although Imps/Exps are overexpressed in a number of transformed cell lines and patient tumor tissues, the efficiency of nucleocytoplasmic transport in transformed cell types compared with nontransformed cells has not been investigated. Here we use quantitative live cell imaging of 3 isogenic nontransformed/transformed cell pairs to show that nuclear accumulation of nuclear localization signal (NLS)-containing proteins, but not their NLS-mutated derivatives, is increased up to 7-fold in MCF10CA1h human epithelial breast carcinoma cells and in simian virus 40 (SV40)-transformed fibroblasts of human and monkey origin, compared with their nontransformed counterparts. The basis for this appears to be a significantly faster rate of nuclear import in transformed cell types, as revealed by analysis using fluorescence recovery after photobleaching for the human MCF10A/MCF10CA1h cell pair. Nuclear accumulation of NLS/nuclear export signal-containing (shuttling) proteins was also enhanced in transformed cell types, experiments using the nuclear export inhibitor leptomycin B demonstrating that efficient Exp-1-mediated nuclear export was not impaired in transformed compared with nontransformed cells. Enhanced nuclear import and export efficiencies were found to correlate with 2- to 4-fold higher expression of specific Imps/Exps in transformed cells, as indicated by quantitative Western blot analysis, with ectopic expression of Imps able to enhance NLS nuclear accumulation levels up to 5-fold in nontransformed MCF10A cells. The findings indicate that transformed cells possess altered nuclear transport properties, most likely due to the overexpression of Imps/Exps. The findings have important implications for the development of tumor-specific drug nanocarriers in anticancer therapy.

Auto-activation of c-JUN Gene by Amino Acid Deprivation of Hepatocellular Carcinoma Cells Reveals a Novel c-JUN-mediated Signaling Pathway

Mammalian cells respond to protein or amino acid (AA) limitation by activating a number of signaling pathways, collectively referred to as the AA response (AAR), that modulate a range of cellular functions, including transcriptional induction of target genes. This study demonstrates that in hepatocellular carcinoma cells, expression of c-JUN, JUN-B, c-FOS, and FOS-B was induced by the AAR, whereas JUN-D, FRA-1, and FRA-2 were not. Of the four activated FOS/JUN members, c-JUN made the largest contribution to the induction of several known AAR target genes. For several human liver, prostate, and ovarian cell lines, the AAR-induced increase in c-JUN expression was greater in transformed cells compared with nontransformed counterparts, an effect independent of cell growth rate. Thus far, the best characterized AA-responsive genes are all transcriptionally activated by ATF4, but the AAR-dependent induction of c-JUN transcription was ATF4-independent. The increased expression of c-JUN was dependent on ATF2 and on activation of the MEK-ERK and JNK arms of the MAPK signaling pathways. Formation of c-JUN-ATF2-activated heterodimers was increased after AA limitation, and c-JUN or ATF2 knockdown suppressed the induction of c-JUN and other AAR target genes. AA deprivation triggers a feed-forward process that involves phosphorylation of existing c-JUN protein by JNK and subsequent auto-activation of the c-JUN gene by recruitment of c-JUN and ATF2 to two AP-1 sites within the proximal promoter. The results document the novel observation that AP-1 sequences within the c-JUN gene can function as transcriptional amino acid-response elements.

Abstract 1066: ERK/RSK and JNK signaling cooperatively mediate oncogenic Ras-induced death receptor 5 expression through enhancing CHOP, Elk1 and c-Jun-dependent transcription

Abstract Oncogenic mutation of Ras frequently occurs in many types of cancers and plays a critical role in cell transformation and oncogenesis. Interestingly, the oncogenic Ras has been documented to induce the expression of death receptor 5 (DR5), a pro-apoptotic death domain-containing cell surface receptor for TRAIL, through currently undefined mechanisms. The current study attempted to address the mechanisms by which the oncogenic Ras induces DR5 expression. Enforced expression of the oncogenic H-Ras or K-Ras (e.g., RasV12) increased DR5 promoter activity and DR5 expression. This was also accompanied by activation of both ERK/RSK and JNK signaling pathways as evidenced by increased levels of p-ERK, p-RSK, p-JNK and p-c-Jun as well as their downstream proteins, CHOP, Elk1 and c-Jun. Gene silencing-mediated inhibition of either ERK, RSK or JNK activation blocked RasV12-induced DR5 expression. Moreover, knockdown of CHOP, Elk1 or c-Jun abrogated RasV12-induced DR5 expression as well. These data collectively indicate that both ERK/RSK and JNK signaling pathways are required for RasV12-induced DR5 expression. Triple gene (CHOP, Elk1 and c-Jun) co-expressed resulted in the highest DR5 promoter transactivation and protein expression in comparison to single or double gene expression. Consistently, oligonucleotide pull-down assay could detect Elk1, CHOP and c-Jun proteins bound to the DR5 promoter in cells co-transfected with CHOP, Elk1 and c-Jun genes or transfected with Ras-V12 gene, indicating that these proteins together bind to the DR5 promoter region. Thus we suggest that these three proteins cooperatively participate in RasV12-induced DR5 expression by enhancing its transcription. Importantly, we found that RasV12-transformed human ovarian cells were more sensitive than their non-transformed counterparts to undergo TRAIL-induced apoptosis. In summary, this study demonstrated that oncogenic Ras induces DR5 expression through activation of both ERK/RSK and JNK signaling pathways and subsequent CHOP, Elk1 and c-Jun-mediated gene transactivation. Cancers with Ras mutation may be suitable for TRAIL-based cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1066. doi:10.1158/1538-7445.AM2011-1066

Expression of anti-sclerotinia scFv in transgenic Brassica napus enhances tolerance against stem rot

Canola is an important agricultural crop imparting a significant contribution to global oilseed production. As such, optimizing yield and quality is of paramount importance and canola production can be significantly affected by sclerotinia stem rot. The utility of recombinant antibody technology in plant protection has been explored by many researchers and shows promise for the generation of new lines of agriculturally significant crops with greater resistance to diseases. The objective of the current study was to generate recombinant pathogen specific antibody (scFv)-expressing transgenic Brassica napus plants with increased tolerance to the phytopathogenic fungus, Sclerotinia sclerotiorum. Transgenic canola (B. napus) lines expressing S. sclerotiorum-specific scFv antibody showed a significant level of tolerance towards S. sclerotiorum as compared to their non-transformed counterparts. Both incidence and progression of S. sclerotiorum-induced disease symptoms were reduced in plants expressing the recombinant scFv.

Sphingolipid rheostat alterations related to transformation can be exploited for specific induction of lysosomal cell death in murine and human glioma

The search for cancer cell-specific targets suffers from a lack of integrative approaches that take into account the relative contributions of several mechanisms or pathways involved in cell death. A systematic experimental and computational comparison of murine glioma cells with astrocytes, their nontransformed counterparts, identified differences in the sphingolipid (SL) rheostat linked to an increased lysosomal instability in glioma cells. In vitro and in silico analyses indicate that sphingosine metabolized in lysosomes was preferentially recycled into ceramide, the prodeath member of the rheostat, in astrocytes. In glioma cells, it preferentially was used for production of the prosurvival sphingosine-1-phosphate (S1P). A combination of tumor necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS), and interferon gamma (IFN-gamma) strongly decreased S1P production that resulted in abnormal lysosome enlargement and cell death associated with mitochondrial dysfunction of glioma cells only. Lack of intracellular S1P in glioma cells was concomitant with protein and lipid accumulation in enlarged lysosomes, indicating a blockade in lysosome recycling, and hence a role for S1P in membrane trafficking. A pharmacological sphingosine kinase inhibitor efficiently replaced the TNF-alpha, LPS, and IFN-gamma combination and killed murine and human glioma cells without affecting astrocytes. Our study provides evidence for a novel mechanism of lysosomal death dependent upon the SL rheostat that can be specifically triggered in glioma cells. It further strengthens the potential of cancer therapies based on specific ceramide pathway alterations.

Enhanced proline accumulation and salt stress tolerance of transgenic indica rice by over-expressing P5CSF129A gene

Δ1-pyrroline-5-carboxylate synthetase (P5CS) is a proline biosynthetic pathway enzyme and is known for conferring enhanced salt and drought stress in transgenics carrying this gene in a variety of plant species; however, the wild-type P5CS is subjected to feedback control. Therefore, in the present study, we used a mutagenized version of this osmoregulatory gene-P5CSF129A, which is not subjected to feedback control, for producing transgenic indica rice plants of cultivar Karjat-3 via Agrobacterium tumefaciens. We have used two types of explants for this purpose, namely mature embryo-derived callus and shoot apices. Various parameters for transformation were optimized including antibiotic concentration for selection, duration of cocultivation, addition of phenolic compound, and bacterial culture density. The resultant primary transgenic plants showed more enhanced proline accumulation than their non-transformed counterparts. This proline level was particularly enhanced in the transgenic plants of next generation (T1) under 150 mM NaCl stress. The higher proline level shown by transgenic plants was associated with better biomass production and growth performance under salt stress and lower extent of lipid peroxidation, indicating that overproduction of proline may have a role in counteracting the negative effect of salt stress and higher maintenance of cellular integrity and basic physiological processes under stress.

Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

The importance of mitogen-activated protein kinase signaling in melanoma is underscored by the prevalence of activating mutations in N-Ras and B-Raf, yet clinical development of inhibitors of this pathway has been largely ineffective, suggesting that alternative oncogenes may also promote melanoma. Notch is an interesting candidate that has only been correlated with melanoma development and progression; a thorough assessment of tumor-initiating effects of activated Notch on human melanocytes would clarify the mounting correlative evidence and perhaps identify a novel target for an otherwise untreatable disease. Analysis of a substantial panel of cell lines and patient lesions showed that Notch activity is significantly higher in melanomas than their nontransformed counterparts. The use of a constitutively active, truncated Notch transgene construct (N(IC)) was exploited to determine if Notch activation is a "driving" event in melanocytic transformation or instead a "passenger" event associated with melanoma progression. N(IC)-infected melanocytes displayed increased proliferative capacity and biological features more reminiscent of melanoma, such as dysregulated cell adhesion and migration. Gene expression analyses supported these observations and aided in the identification of MCAM, an adhesion molecule associated with acquisition of the malignant phenotype, as a direct target of Notch transactivation. N(IC)-positive melanocytes grew at clonal density, proliferated in limiting media conditions, and also exhibited anchorage-independent growth, suggesting that Notch alone is a transforming oncogene in human melanocytes, a phenomenon not previously described for any melanoma oncogene. This new information yields valuable insight into the basic epidemiology of melanoma and launches a realm of possibilities for drug intervention in this deadly disease.

Transcriptional and functional defects of dendritic cells derived from the MUTZ‐3 leukaemia line

Dendritic cells (DC) generated from MUTZ-3, an immortalized acute myeloid leukaemia-derived cell line, have potential application as a model for the study of human DC, and as a tool with which to stimulate immunotherapeutic responses to cancer. However, the relationship of MUTZ-3 DC to their non-transformed counterparts remains incompletely understood. Immunoselected CD14+ MUTZ-3 cells were used to generate a homogeneous population of DC (M3DC). These cells had a cell surface phentoype and morphology characteristic of conventional monocyte-derived DC (MDDC). Whole genome transcriptome comparison of M3DC and MDDC however, revealed extensive differences between these two cell types. Functional ontology-based data analysis revealed three enriched clusters of genes downregulated in M3DC, with functions in pathogen recognition, DC maturation and cytokine/chemokine signalling. Downregulation of protein expression was confirmed for several of these genes. The molecular differences were accompanied by a profoundly impaired phenotypic and functional response of M3DC to microbial stimulation. The immortalized phenotype of MUTZ-3 therefore reflects not only deregulated proliferative capacity, but substantial perturbation of normal antigen-presenting cell function. These results have important implications for studies using MUTZ-3 as a model of MDDC or for cancer immunotherapy.

Central nervous system involvement in patients with mycosis fungoides and cutaneous large-cell transformation

Central nervous system (CNS) involvement in mycosis fungoides (MF) is a rare occurrence with devastating consequences. Large-cell transformation (LCT) of MF is associated with a poor prognosis and increased extracutaneous manifestations. In this report, we present 5 patients with MF, LCT, and CNS disease to better define their presentation, treatment, and prognosis and present critical differences they have compared with their nontransformed counterparts. We found that patients with LCT and MF are at increased risk of CNS involvement. In addition, whereas patients with nontransformed MF typically have significant skin and visceral involvement at the time of CNS disease, some patients with LCT have no extracutaneous symptoms and their skin MF is in remission at the time of CNS presentation. Transformed patients tend to present with speech and visual difficulties secondary to mass lesions. Therefore, we recommend at least annual screening of patients with transformed MF for CNS disease with head computerized tomography with orbital cuts.

Global Impact of Oncogenic Src on a Phosphotyrosine Proteome

Elevated activity of Src, the first characterized protein-tyrosine kinase, is associated with progression of many human cancers, and Src has attracted interest as a therapeutic target. Src is known to act in various receptor signaling systems to impact cell behavior, yet it remains likely that the spectrum of Src protein substrates relevant to cancer is incompletely understood. To better understand the cellular impact of deregulated Src kinase activity, we extensively applied a mass spectrometry shotgun phosphotyrosine (pTyr) proteomics strategy to obtain global pTyr profiles of Src-transformed mouse fibroblasts as well as their nontransformed counterparts. A total of 867 peptides representing 563 distinct pTyr sites on 374 different proteins were identified from the Src-transformed cells, while 514 peptides representing 275 pTyr sites on 167 proteins were identified from nontransformed cells. Distinct characteristics of the two profiles were revealed by spectral counting, indicative of pTyr site relative abundance, and by complementary quantitative analysis using stable isotope labeling with amino acids in cell culture (SILAC). While both pTyr profiles are replete with sites on signaling and adhesion/cytoskeletal regulatory proteins, the Src-transformed profile is more diverse with enrichment in sites on metabolic enzymes and RNA and protein synthesis and processing machinery. Forty-three pTyr sites (32 proteins) are predicted as major biologically relevant Src targets on the basis of frequent identification in both cell populations. This select group, of particular interest as diagnostic biomarkers, includes well-established Src sites on signaling/adhesion/cytoskeletal proteins, but also uncharacterized sites of potential relevance to the transformed cell phenotype.