Proteomic analysis of gemcitabine-induced drug resistance in pancreatic cancer cells

Currently, the most effective agent against pancreatic cancer is gemcitabine (GEM), which inhibits tumor growth by interfering with DNA replication and blocking DNA synthesis. However, GEM-induced drug resistance in pancreatic cancer compromises the therapeutic efficacy of GEM. To investigate the molecular mechanisms associated with GEM-induced resistance, 2D-DIGE and MALDI-TOF mass spectrometry were performed to compare the proteomic alterations of a panel of differential GEM-resistant PANC-1 cells with GEM-sensitive pancreatic cells. The proteomic results demonstrated that 33 proteins were differentially expressed between GEM-sensitive and GEM-resistant pancreatic cells. Of these, 22 proteins were shown to be resistance-specific and dose-dependent in the regulation of GEM. Proteomic analysis also revealed that proteins involved in biosynthesis and detoxification are significantly over-expressed in GEM-resistant PANC-1 cells. In contrast, proteins involved in vascular transport, bimolecular decomposition, and calcium-dependent signal regulation are significantly over-expressed in GEM-sensitive PANC-1 cells. Notably, both protein-protein interaction of the identified proteins with bioinformatic analysis and immunoblotting results showed that the GEM-induced pancreatic cell resistance might interplay with tumor suppressor protein p53. Our approach has been shown here to be useful for confidently detecting pancreatic proteins with differential resistance to GEM. Such proteins may be functionally involved in the mechanism of chemotherapy-induced resistance.     

Targeting ASCT2-mediated glutamine metabolism inhibits proliferation and promotes apoptosis of pancreatic cancer cells

Some tumor cells have a high rate of glutamine uptake and exhibit glutamine addiction. Alanine-serine cysteine-preferring transporter 2 (ASCT2) is a major mediator of glutamine supply in many tumor cells, but the underlying effects and mechanisms of ASCT2 in pancreatic cancer (PC) are largely unknown. Our results show that ASCT2 expression is significantly higher in PC than in normal pancreatic duct cells and pancreas. Utilizing the Kaplan–Meier Plotter database, a high expression of SLC1A5 mRNA was significantly associated with poor overall survival (OS) in patients with PC. shRNA-mediated inhibition of ASCT2 function in vitro can significantly decrease glutamine consumption, α-ketoglutarate (α-KG) production and ATP generation and increase the reactive oxygen species (ROS) level. Moreover, the antioxidant N-acetylcysteine partially attenuated the increase in the ROS levels and reduced ATP generation. These data suggest that ASCT2 mediates glutamine metabolism and maintains redox homeostasis in PC. To further investigate whether ASCT2 is involved in PC cell growth, we blocked ASCT2 activity with the ASCT2 inhibitor l-γ-glutamyl-p-nitroanilide (GPNA) and silenced the expression of ASCT2 with specific shRNAs. We found that the growth of PC cells was significantly inhibited. Additionally, knockdown of ASCT2 induced apoptosis through the Akt/mTOR signaling pathway. Furthermore, the loss of ASCT2 in BxPC-3 cell xenografts significantly inhibited tumor growth in vivo, and this effect was associated with an increase in cleaved caspase-3 expression and a decrease in Ki67 staining. Taken together, our results show that ASCT2 may be utilized as a putative therapeutic target for PC.     

Lactate promotes glutamine uptake and metabolism in oxidative cancer cells

Oxygenated cancer cells have a high metabolic plasticity as they can use glucose, glutamine and lactate as main substrates to support their bioenergetic and biosynthetic activities. Metabolic optimization requires integration. While glycolysis and glutaminolysis can cooperate to support cellular proliferation, oxidative lactate metabolism opposes glycolysis in oxidative cancer cells engaged in a symbiotic relation with their hypoxic/glycolytic neighbors. However, little is known concerning the relationship between oxidative lactate metabolism and glutamine metabolism. Using SiHa and HeLa human cancer cells, this study reports that intracellular lactate signaling promotes glutamine uptake and metabolism in oxidative cancer cells. It depends on the uptake of extracellular lactate by monocarboxylate transporter 1 (MCT1). Lactate first stabilizes hypoxia-inducible factor-2α (HIF-2α), and HIF-2α then transactivates c-Myc in a pathway that mimics a response to hypoxia. Consequently, lactate-induced c-Myc activation triggers the expression of glutamine transporter ASCT2 and of glutaminase 1 (GLS1), resulting in improved glutamine uptake and catabolism. Elucidation of this metabolic dependence could be of therapeutic interest. First, inhibitors of lactate uptake targeting MCT1 are currently entering clinical trials. They have the potential to indirectly repress glutaminolysis. Second, in oxidative cancer cells, resistance to glutaminolysis inhibition could arise from compensation by oxidative lactate metabolism and increased lactate signaling.     

Proteomic analysis of cancer stem cells in human prostate cancer cells

Results from recent studies support the hypothesis that cancer stem cells (CSCs) are responsible for tumor initiation and formation. Here, we applied a proteome profiling approach to investigate the mechanisms of CSCs and to identify potential biomarkers in the prostate cancer cell line DU145. Using MACS, the DU145 prostate cancer cell line was isolated into CD44+ or CD44− cells. In sphere culture, CD44+ cells possessed stem cell characteristics and highly expressed genes known to be important in stem cell maintenance. In addition, they showed strong tumorigenic potential in the clonogenic assay and soft agar colony formation assay. We then analyzed and identified proteins that were differentially expressed between CD44+ and CD44− using two-dimensional gel electrophoresis and LC-MS/MS. Cofilin and Annexin A5, which are associated with proliferation or metastasis in cancer, were found to be positively correlated with CD44 expression. These results provide information that will be important to the development of new cancer diagnostic tools and understanding the mechanisms of CSCs although a more detailed study is necessary to investigate the roles of Cofilin and Annexin A5 in CSCs.     

Proteomic analysis reveals a metabolism shift in a laboratory fluconazole-resistant Candida albicans strain

Multifactorial and multistep alterations are involved in acquired fluconazole (FLC) resistance in Candida albicans. In this study, a FLC-resistant C. albicans strain was obtained by serial cultures of a FLC-susceptible C. albicans strain in incrementally increasing concentrations of FLC. The comparative proteomic study, confirmed by real-time RT-PCR, was performed with the susceptible parental strain and the resistant daughter strain to identify proteins altered during the development of FLC resistance. Our analysis of the differentially expressed proteins identified 22 different proteins, most of which were related to energy metabolisms (e.g., Pgk1, Fba1, and Adh1), and some of which have been previously identified as being involved in FLC resistance in C. albicans (e.g., Ald5, Cdc19, and Gap1). Functional analysis revealed lower intracellular ATP level and mitochondrial membrane potential, less endogenous reactive oxygen species generation in response to antifungal agents, and identical susceptibility to exogenous hydrogen peroxide, heat, and hyperosmotic shock in the resistant strain compared with the susceptible strain. Our results suggest that a metabolism shift might contribute to FLC resistance in C. albicans.     

Proteomic changes in renal cancer and co-ordinate demonstration of both the glycolytic and mitochondrial aspects of the Warburg effect

Renal cell carcinoma (RCC) is the tenth most common cancer although the incidence is increasing. The main clinical problems stem from the relatively late presentation of many patients due to the often asymptomatic nature of the illness, and the relative insensitivity of metastatic disease to conventional chemotherapy and radiotherapy. Despite increasing knowledge of some of the genetic changes underlying sporadic renal cancer such as those involving the Von Hippel Lindau (VHL) gene, many of the underlying pathophysiological changes are ill-defined and there remains a need for the identification of disease markers for use in diagnosis and prognosis or as potential therapeutic targets. This study has used a proteomic approach, based on two-dimensional gel electrophoresis and mass spectrometry, to compare the protein profiles of conventional RCC tissue with patient-matched normal kidney cortex. Sequencing of 32 protein spots with significantly increased expression in RCC samples (>/= 4/6 patients) and 41 proteins whose levels decreased (6/6 patients) confirmed several previously known RCC-associated changes such as increases in Mn-superoxide dismutase, lactate dehydrogenase-A, aldolase A and C, pyruvate kinase M2, and thymidine phosphorylase. Additionally, several previously unknown changes were identified, including increased expression of three members of the annexin family and increased levels of the actin depolymerisation factor cofilin. The Warburg effect was also demonstrated with the identification of increases in proteins involved in the majority of steps in the glycolytic pathway and decreases in the gluconeogenic reactions, together with a parallel decrease in several mitochondrial enzymes. A number of the alterations seen were further confirmed in additional samples by immunohistochemistry, Western blotting, and laser capture microdissection.     

Comprehensive analysis of cellular galectin-3 reveals no consistent oncogenic function in pancreatic cancer cells

Galectin-3 (Gal-3), a 31 kDa member of the family of beta-galactoside-binding proteins, has been implicated in the progression of different human cancers. However, the proposed roles differ widely, ranging from tumor-promoting cellular functions and negative impact on patient prognosis to tumor-suppressive properties and positive prognostic impact. We and others have previously identified Gal-3 as overexpressed in pancreatic cancer as compared to chronic pancreatitis and normal pancreatic tissue. The purpose of this study was thus the comprehensive analysis of putative cellular functions of Gal-3 by transient as well as stable silencing or overexpression of Gal-3 in a panel of 6 well-established pancreatic cancer cell lines. Our results confirm that galectin-3 is upregulated at the mRNA level in pancreatic cancer and strongly expressed in the majority of pancreatic cancer cell lines. In individual cell lines, transient knockdown of Gal-3 expression resulted in moderate inhibitory effects on proliferation, migration or anchorage-independent growth of the cells, but these effects were not consistent across the spectrum of analyzed cell lines. Moreover, functional effects of the modulation of Gal-3 expression were not observed in stable knockdown or overexpression approaches in vitro and did not alter the growth characteristics of nude mouse xenograft tumors in vivo. Our data thus do not support a direct functional role of Gal-3 in the malignant transformation of pancreatic epithelial cells, although paracrine or systemic effects of Gal-3 expression are not excluded.     

Lobetyolin induces apoptosis of colon cancer cells by inhibiting glutamine metabolism

The purpose of the present study was to evaluate the anti-cancer property of Lobetyolin on colorectal cancer and explore its potential mechanism. Lobetyolin was incubated with HCT-116 cells in the absence or presence of ASCT2 inhibitor Benser or p53 inhibitor Pifithrin-α. The levels of glutamine, glutamic acid, α-ketoglutarate, ATP and GSH were determined to measure the glutamine metabolism. Annexin V-FITC/PI staining and TUNEL assay were applied to estimate the apoptotic condition. The levels of ASCT2 were examined by RT-qPCR, Western blot and immunofluorescence staining. The expressions of cleaved-caspase-3, caspase-3, cleaved-caspase-7, caspase-7, cleaved-PARP, PARP, p53, p21, bax and survivin were detected using Western blot analysis. As a result, the treatment with Lobetyolin effectively induced apoptosis and glutamine metabolism in HCT-116 cells through ASCT2 signalling. The inhibition of ASCT2 reduced the glutamine-related biomarkers and augmented the apoptotic process. We further found that the effect of Lobetyolin on HCT-116 was related to the expressions of p21 and bax, and transportation of p53 to nucleus. The inhibition of p53 by Pifithrin-α promoted the inhibitory effect of Lobetyolin on ASCT2-mediated apoptosis. Lobetyolin also exerted anti-cancer property in nude mice. In conclusion, the present work suggested that Lobetyolin could induce the apoptosis via the inhibition of ASCT2-mediated glutamine metabolism, which was possibly governed by p53.     

The Warburg effect and its cancer therapeutic implications

Increased aerobic glycolysis in cancer, a phenomenon known as the Warburg effect, has been observed in various tumor cells and represents a major biochemical alteration associated with malignant transformation. Although the exact molecular mechanisms underlying this metabolic change remain to be elucidated, the profound biochemical alteration in cancer cell energy metabolism provides exciting opportunities for the development of therapeutic strategies to preferentially kill cancer cells by targeting the glycolytic pathway. Several small molecules capable of inhibiting glycolysis in experimental systems have been shown to have promising anticancer activity in vitro and in vivo. This review article provides a brief summary of our current understanding of the Warburg effect, the underlying mechanisms, and its influence on the development of therapeutic strategies for cancer treatment.     

Proteomic and redox-proteomic analysis of berberine-induced cytotoxicity in breast cancer cells

Berberine is a natural product isolated from herbal plants such as Rhizoma coptidis which has been shown to have anti-neoplastic properties. However, the effects of berberine on the behavior of breast cancers are largely unknown. To determine if berberine might be useful in the treatment of breast cancer and its cytotoxic mechanism, we analyzed the impact of berberine treatment on differential protein expression and redox regulation in human breast cancer cell line MCF-7 using lysine- and cysteine-labeling two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS). This study demonstrated that 96 and 22 protein features were significantly changed in protein expression and thiol reactivity, respectively and revealed that berberine-induced cytotoxicity in breast cancer cells involves dysregulation of protein folding, proteolysis, redox regulation, protein trafficking, cell signaling, electron transport, metabolism and centrosomal structure. Our work shows that this combined proteomic strategy provides a rapid method to study the molecular mechanisms of berberine-induced cytotoxicity in breast cancer cells. The identified targets may be useful for further evaluation as potential targets in breast cancer therapy.     

Proteomic analysis of colorectal cancer reveals alterations in metabolic pathways: mechanism of tumorigenesis

Colorectal cancer is the second leading killer cancer worldwide and presently the most common cancer among males in Singapore. The study aimed to detect changes of protein profiles associated with the process of colorectal tumorigenesis to identify specific protein markers for early colorectal cancer detection and diagnosis or as potential therapeutic targets. Seven pairs of colorectal cancer tissues and adjacent normal mucosa were examined by two-dimensional gel electrophoresis at basic pH range (pH 7-10). Intensity changes of 34 spots were detected with statistical significance. 16 of the 34 spots were identified by MALDI-TOF/TOF tandem mass spectrometry. Changes in protein expression levels revealed a significantly enhanced glycolytic pathway (Warburg effect), a decreased gluconeogenesis, a suppressed glucuronic acid pathway, and an impaired tricarboxylic acid cycle. Observed changes in protein abundance were verified by two-dimensional DIGE. These changes reveal an underlying mechanism of colorectal tumorigenesis in which the roles of impaired tricarboxylic acid cycle and the Warburg effect may be critical.     

Nitric oxide is a positive regulator of the Warburg effect in ovarian cancer cells

Ovarian cancer (OVCA) is among the most lethal gynecological cancers leading to high mortality rates among women. Increasing evidence indicate that cancer cells undergo metabolic transformation during tumorigenesis and growth through nutrients and growth factors available in tumor microenvironment. This altered metabolic rewiring further enhances tumor progression. Recent studies have begun to unravel the role of amino acids in the tumor microenvironment on the proliferation of cancer cells. One critically important, yet often overlooked, component to tumor growth is the metabolic reprogramming of nitric oxide (NO) pathways in cancer cells. Multiple lines of evidence support the link between NO and tumor growth in some cancers, including pancreas, breast and ovarian. However, the multifaceted role of NO in the metabolism of OVCA is unclear and direct demonstration of NO''s role in modulating OVCA cells'' metabolism is lacking. This study aims at indentifying the mechanistic links between NO and OVCA metabolism. We uncover a role of NO in modulating OVCA metabolism: NO positively regulates the Warburg effect, which postulates increased glycolysis along with reduced mitochondrial activity under aerobic conditions in cancer cells. Through both NO synthesis inhibition (using L-arginine deprivation, arginine is a substrate for NO synthase (NOS), which catalyzes NO synthesis; using L-Name, a NOS inhibitor) and NO donor (using DETA-NONOate) analysis, we show that NO not only positively regulates tumor growth but also inhibits mitochondrial respiration in OVCA cells, shifting these cells towards glycolysis to maintain their ATP production. Additionally, NO led to an increase in TCA cycle flux and glutaminolysis, suggesting that NO decreases ROS levels by increasing NADPH and glutathione levels. Our results place NO as a central player in the metabolism of OVCA cells. Understanding the effects of NO on cancer cell metabolism can lead to the development of NO targeting drugs for OVCAs.Despite recent medical and pharmaceutical advances in cancer research, ovarian cancer (OVCA) remains one of the most deadly gynecological malignancies, with most of the cancer first detected in late stages when metastasis has already occurred.¹ Only 20% of OVCA patients are diagnosed when cancer has not spread past the ovaries; in the other 80% of cases, the cancer has metastasized, most frequently to the peritoneum.² Platinum-based preoperative chemotherapy is the standard of care of early stage disease, and surgical resection along with platinum-based postoperative chemotherapy is the standard of care for late stage disease.¹ However, many platinum-based chemotherapy drugs come with unwanted side effects. Therefore, an alternative therapy for OVCA is needed.Nitric oxide (NO) shows promise either as a cancer therapeutic agent by itself or as a target of cancer therapies.³ This may be because NO can act as a signaling molecule or as a source of oxidative and nitrosative stress.⁴ NO can stimulate mitochondrial biogenesis through PGC-1-related coactivator⁵ and increase mitochondrial function.^{6, 7} In follicular thyroid carcinoma cells, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, was shown to increase the expression of genes involved in mitochondrial biogenesis.^{8, 9} A 14-day treatment of lung carcinoma cells with dipropylenetriamine NONOate (DETA-NONOate), another NO donor, increased cell migration compared with the absence of treatment.¹⁰ In breast cancer cells, exogenous NO increased cell proliferation, as well as cyclin-D1 and ornithine decarboxylase expression.¹¹ In prostate cancer cells, NO was shown to inhibit androgen receptor-dependent promoter activity and proliferation of androgen-dependent cells, indicating that NO would select for the development of prostate cancer cells that are androgen-independent.¹² NO has even been shown to inhibit mitochondrial ATP production, and therefore inhibit apoptosis, as ATP is necessary for the apoptotic process.¹³ Moreover, inducible nitric oxide synthase (iNOS) knockout mice had less tumor formation than wild-type mice, indicating that NO promotes lung tumorigenesis.¹⁴ On the other hand, NO production, as induced by proinflammatory cytokines, induced apoptosis in OVCA cells.³ NOS overexpression by transfection of a plasmid containing NOS-3 DNA resulted in increased cell death in HepG2 cells.¹⁵ In another study, NO was implicated in N-(4-hydroxyphenyl) retinamide-mediated apoptosis.¹⁶ Finally, iNOS expression in p53-depleted mice increased apoptosis of lymphoma cells compared with p53-deficient mice without iNOS expression.¹⁷ Therefore, NO has been seen to have both an anti-tumorigenic as well as a pro-tumorigenic effect.Arginine, a conditionally essential amino acid used to produce NO, is also a potential target for cancer therapy. L-arginine is normally produced by the body; however, in some diseased states, more arginine than what the body normally produces is required.¹⁸ Arginine sources include protein breakdown or directly from the diet, in addition to de novo synthesis.¹⁹ In the de novo production of L-arginine, citrulline and aspartate are first converted to argininosuccinate by arginase, which is then split into arginine and fumarate by argininosuccinate lyase.²⁰ L-arginine can also be converted to citrulline and NO through NO synthase (NOS).¹⁹ Some cancer cells, including melanoma and hepatocellular carcinoma, do not express argininosuccinate synthase (ASS), an enzyme involved in arginine production and thus rely on exogenous arginine.¹⁹ For these cancers, arginine-deprivation therapy is being heavily explored as a treatment.^{21, 22} OVCA cells have been shown to express ASS.²³ In fact, OVCA cells were shown to have increased expression of ASS compared with normal ovarian surface epithelium.²⁴ As OVCA can synthesize arginine de novo, strategies which target arginine''s conversion into citrulline are needed for regulating OVCA tumor growth.Recent studies suggest that cancer cells undergo metabolic reprogramming, which drives cancer cells'' growth and progression.^{25, 26, 27, 28, 29, 30, 31, 32, 33} One critically important, yet often overlooked, component to tumor growth is the metabolic rewiring of NO pathways in OVCA cells. Despite considerable investigation on NO''s regulation of cancer cell proliferation and growth, mechanistic details regarding the effect of NO on cancer cell metabolism is still lacking: specifically, how NO affects glycolysis, TCA cycle flux, and ROS production. Studies on the effects of NO on cancer cell metabolism have mainly focused on the effect of NO on mitochondrial respiration.^{34, 35, 36, 37} NO has been shown to inhibit cytochrome c oxidase (COX) in the mitochondria of breast cancer cells, as well as decrease oxygen consumption rate.^{37, 38, 39} Moncada and colleagues studied the effect of NO on the metabolism of rat cortical astrocytes and neurons, two cells with different glycolytic capacities. They showed that NO decreased ATP concentration, which led to an increase in glycolysis in astrocytes, but not in neurons, indicating that glycolytic capacity affects the metabolic response of these cells to NO.⁴⁰ NO was shown to reduce ATP production via OXPHOS in rat reticulocytes, cells that produce 90% of their ATP from OXPHOS.⁴¹ Endothelial NOS (eNOS) was shown to have a role in the upregulation of GLUT4 transporters by AMPK and AICAR in the heart muscle.⁴² Additionally, NO can serve to stabilize HIF-1α in hypoxic conditions through S-nitrosylation of PHD2,⁴ and as HIF-1α upregulates GLUT transporters and glycolysis,⁴³ NO may affect the metabolism of cancer cells.Although NO is found to affect glycolysis of normal cells, how NO modulates glycolysis of OVCA cells is less understood. The multifaceted role of NO in the metabolism of OVCA is unclear, and direct demonstration of NO''s role in modulating the metabolism of OVCA cells is lacking. This study aims at understanding the mechanistic links between NO and the overall cancer metabolism – specifically, its effects on glycolysis, TCA cycle, OXPHOS, and ROS production – of OVCA cells. Our results show that NO decreases mitochondrial respiration, forcing OVCA cells to undergo higher glycolytic rates to maintain ATP production levels. Our work is the first to illustrate the central role of NO on OVCA metabolism – specifically, showing how NO (i) positively regulates the Warburg effect in OVCA cell, (ii) maintains low ROS levels by upregulating NADPH generation, and (ii) negatively alters mitochondrial respiration, thus promoting cancer growth and proliferation. Our work is also unique in that it is the first to explore the effects of NO on TCA cycle flux and glutaminolysis, potentially also affecting ROS levels by affecting antioxidant levels. In conclusion, by elucidating the effects of NO on cancer metabolism and ROS levels, we have a better understanding of the different mechanisms by which NO affects cancer cell growth. This understanding may lead to potentially useful therapies to halt cancer progression.     

Proteomic analysis reveals the spatial heterogeneity of immobilized Taxus cuspidata cells in support matrices

A proteomic approach was used to study the responses of Taxus cuspidata cells to local microenvironments in different zones of immobilized support matrices. Analysis of protein spots by 2-DE revealed significant differences in the abundance of 31 spots, 28 spots, and 23 spots in outer, middle, and central zone cells between the immobilized and suspended cells. Six of these proteins, identified by MALDI-TOF-MS, were involved in the regulation of carbohydrate, nitrogen, and sulfur metabolisms. Immobilization triggered an increase in taxol production of the immobilized cells in the middle and central zones compared to that of the suspended cells. A negative relation between taxol production and the mitotic index was observed in the cells in the immobilization support matrix. Cells in the outer zone had high mitotic index and low taxol production, while cells in the middle and central zones showed low mitotic index and high taxol production. The abundance of S-adenosylmethionine synthetase, which was identified as one of the differentially expressed proteins, was positively correlated to the cell division activity in the immobilized cell cultures.     

Proteomic analysis reveals the differential histone programs between male germline cells and vegetative cells in Lilium davidii

In flowering plants, male germline fate is determined after asymmetric division of the haploid microspore. Daughter cells have distinct fates: the generative cell (GC) undergoes further mitosis to generate sperm cells (SCs), and the vegetative cell (VC) terminally differentiates. However, our understanding of the mechanisms underlying germline development remains limited. Histone variants and modifications define chromatin states, and contribute to establishing and maintaining cell identities by affecting gene expression. Here, we constructed a lily protein database, then extracted and detailed histone entries into a comprehensive lily histone database. We isolated large amounts of nuclei from VCs, GCs and SCs from lily, and profiled histone variants of all five histone families in all three cell types using proteomics approaches. We revealed 92 identities representing 32 histone variants: six for H1, 11 for H2A, eight for H2B, five for H3 and two for H4. Nine variants, including five H1, two H2B, one H3 and one H4 variant, specifically accumulated in GCs and SCs. We also detected H3 modification patterns in the three cell types. GCs and SCs had almost identical histone profiles and similar H3 modification patterns, which were significantly different from those of VCs. Our study also revealed the presence of multiple isoforms, and differential expression patterns between isoforms of a variant. The results suggest that differential histone programs between the germline and companion VCs may be established following the asymmetric division, and are important for identity establishment and differentiation of the male germline as well as the VC.     

Uptake and metabolism of glutamine in cultured kidney cells

The metabolism of glutamine was investigated in cultured rat kidney cells. Glutamine utilization and product formation were followed as a function of time at either 10 microM or 1 mM initial glutamine concentration. At 1 mM glutamine, glutamate and gamma-glutamylglutamate were the major products formed at the end of a 5-min incubation period; glutamate accounted for 46% while gamma-glutamylglutamate accounted for 33% of the glutamine utilized. With time, glutamate continued to accumulate while gamma-glutamyl peptide formation leveled off. The role of gamma-glutamyl transpeptidase was assessed by using hippurate, a physiological activator of gamma-glutamyl transpeptidase and acivicin, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, an inhibitor of gamma-glutamyl transpeptidase. Hippurate, 4 mM, increased the utilization of glutamine and the formation of glutamate, gamma-glutamyl peptides and ammonia. Exposure of cells to acivicin resulted in 98% inhibition of gamma-glutamyl transpeptidase without effecting phosphate-dependent glutaminase activity. Acivicin inhibition resulted in a decreased utilization of glutamine and product formation as compared to control; 5-oxoproline appearance fell 70%. The fractional distribution of glutamine carbon and nitrogen into its metabolic products in control, hippurate and acivicin-treated cells showed no change at the end of 60 min. The data provide evidence that gamma-glutamyl transpeptidase utilizes glutamine and forms gamma-glutamyl peptides in cultured kidney cells.     

Proteomic technologies and their application to pancreatic cancer

Pancreatic ductal adenocarcinoma is a devastating disease that represents an important health problem. It spreads rapidly at a time when patients have relatively few symptoms and consequently is often only detected at an advanced stage when treatment options are limited. Rapid developments in technology and bioinformatics have recently led to a surge in proteomics-based cancer research. Comparative analysis of protein profiles from nonmalignant and malignant pancreas cells or tissue, or from different stages of pancreatic cancer, potentially offer unique insight into the biology of this tumor type. Furthermore, proteomic approaches may provide novel diagnostic or therapeutic markers for this disease. Although such analyses are still in their infancy, they show great potential in the ongoing battle against this dismal disease.