Effect Of Serum Deprivation Research Articles

Abstract 91: Classification of cancer cells based on unexpected variation in levels and dynamics of pRb, pERK, and pAKT in response to cell synchronization from nutrient deprivation

Abstract The tumor suppressor Rb and the RAS-RAF-MEK-ERK and PI3K-AKT-mTOR signaling cascades are often dysregulated in cancer. As insufficient vasculature in the tumor microenvironment (TME) leads to nutrient deficiency, cellular responses to nutrient deprivation is relevant to consider in the treatment of solid tumors. Here, we demonstrate that levels of pRb, pERK, and pAKT after cell synchronization via nutrient deprivation and after release via serum stimulation vary unexpectedly across a panel of nine cancer and two normal cell lines. From these results, we describe a novel classification of tumor cells defined by their behavior in nutrient-limiting conditions. Four colorectal, four lung, and one breast cancer cell line, and two normal lung fibroblasts were included in the panel. A range of KRAS, NRAS, BRAF, and PIK3CA mutations as well as wild-type cells were represented. In 6-well plates, 1E5-5E5 cells were plated per well in media containing 10% serum, incubated for 16-24 hours, then were nutrient-deprived in media containing 0% or 1% serum for 2, 4, 8, 12, 24, 48, 72, or 96 hours. Synchronization was released by serum stimulation with 20% serum-containing media for 24-48 hours. Cells were imaged, counted, and harvested, then protein levels of Rb, pRb, ERK, pERK, AKT, and pAKT were measured in cell lysates using a western blot. Nutrient deprivation slowed the proliferation of each cell line. Surprisingly, phosphorylation of Rb at Ser795 and Ser807/811 after nutrient deprivation varied across cell lines. pRb remained at baseline in MCF7 cells, decreased in RKO and H1299 cells, and increased in HCT116 cells. Despite this, levels of the pRb upstream regulator CDK6 decreased consistently across these cell lines, suggesting possible alternate regulation of pRb in MCF7 and HCT116 cells. Unanticipated variation in pERK levels after nutrient deprivation was also observed, including a dramatic increase in pERK upon starvation in HCT116 cells but not in others. Similar notable findings were made as far as pAKT. pAKT levels changed dynamically across time points in some cell lines, but consistently increased in some (MCF7) and decreased in others (RKO). Cell synchronization release via serum stimulation caused varying levels of pRb and pERK restoration. Our novel findings reveal a broad new classification of tumor cells defined by their behavior nutrient-limiting conditions. Such classification may guide the use of nutrient deprivation as a predictive biomarker for CDK, MEK, or mTOR inhibitors in solid tumors with nutrient-poor TMEs, as we are currently exploring. We are also evaluating the impact of nutrient deprivation on immune cell killing and cytokine secretion of specific classes of cancer cells that we define here, and are working to establish real-time cell cycle markers to visualize differential effects of serum deprivation and stimulation. Citation Format: Lindsey Carlsen, Wafik S. El-Deiry. Classification of cancer cells based on unexpected variation in levels and dynamics of pRb, pERK, and pAKT in response to cell synchronization from nutrient deprivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 91.

Abstract 3241: IC50-Seeding Density Slope (ISDS)-measurement as a standardized methodology for assessing anti-cancer therapeutic activity in vitro

Abstract The 50% inhibitory concentration (IC50) is a widely used measure of how effective a given anti-cancer therapeutic is at reducing cancer cell viability in vitro. It is known that IC50 of a given therapeutic agent is dependent on the seeding density of the treated cells (in that more densely-seeded cells tend to be more resistant to therapeutic agents). We performed experiments using the CellTiter Glo cell viability assay to assess the cytotoxicity of various chemotherapeutic drugs towards various cancer cell lines. To elucidate the role the extracellular environment of densely-seeded cells plays in cancer cell chemoresistance, we tested conditioned media from cells cultured at high density on cells seeded at low densities. The resulting impact on the IC50 of the low-density cells was determined. The IC50 of the various therapeutic agents tested was cell density-dependent in each cancer cell line examined. Based on this analysis, we propose the IC50-seeding density slope (ISDS), which relates seeding density to the effectiveness of a drug and that we believe could be a standardized quantitative measurement of how a given cancer cell line responds to a given therapeutic treatment. Our results from the conditioned media experiments consistently indicated that the environment of densely-seeded tumor cells conferred chemoresistance to sparsely-seeded cells, suggesting that extracellular cytokines and growth factors may be involved in density-dependent chemoresistance mechanisms. We are currently exploring the effect of serum deprivation on in vitro chemoresistance, in order to further understand how the presence of extracellular nutrients impacts observed IC50 trends. In addition, we are examining the role that cell-cell contact, integrin and FGF signaling pathways play in giving rise to density-dependent chemoresistance. Moreover, to analyze the manner in which conditioned media gives rise to chemoresistance and how this effect relates to intracellular survival pathways, we are performing cytokine profiling experiments. Overall, we describe density-dependent IC50 variations as a hallmark of cancer cells and propose that ISDS may serve as a standardized method of assessing chemosensitivity. Citation Format: Ujwal Punyamurtula, Shengliang Zhang, Thomas Brown, Arielle De La Cruz, Alexander Raufi, Jillian Strandberg, Lindsey Carlsen, Lanlan Zhou, Wafik El-Deiry. IC50-Seeding Density Slope (ISDS)-measurement as a standardized methodology for assessing anti-cancer therapeutic activity in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3241.

Glucose and Serum Deprivation Led to Altered Proliferation, Differentiation Potential and AMPK Activation in Stem Cells from Human Deciduous Tooth.

Stem cell therapy is an evolving treatment strategy in regenerative medicine. Recent studies report stem cells from human exfoliated deciduous teeth could complement the traditional mesenchymal stem cell sources. Stem cells from human exfoliated deciduous teeth exhibit mesenchymal characteristics with multilineage differentiation potential. Mesenchymal stem cells are widely investigated for cell therapy and disease modeling. Although many research are being conducted to address the challenges of mesenchymal stem cell therapy in clinics, most of the studies are still in infancy. Host cell microenvironment is one of the major factors affecting the homing of transplanted stem cell and understanding the factors affecting the fate of stem cells of prime important. In this study we aimed to understand the effects of serum deprivation in stem cells derived from human deciduous tooth. Our study aimed to understand the morphological, transcriptional, cell cycle and stemness based changes of stem cells in nutrient deprived medium. Our results suggest that stem cells in nutrient deprived media undergo low proliferation, high apoptosis and changed the differentiation potential of the stem cells. Serum deprived mesenchymal stem cells exhibited enhanced chondrogenic differentiation potential and reduced osteogenic differentiation potential. Moreover, the activation of key metabolic sensor AMP-activated kinase (AMPK) leads to activation of transcription factors such as FOXO3, which leads to an S phase quiescence. Serum deprivation also enhanced the expression of stemness related genes Sox2 and c-Myc.

Identifying connexin expression and determining gap junction intercellular communication in rainbow trout cells.

Gap junctions are groups of membrane-bound channels that allow the passage of small molecules and ions between cells, permitting cell-cell communication. Because of their importance in cell homeostasis, gap junction presence and function were characterized in three commonly studied rainbow trout cell lines, namely RTgill-W1, RTgutGC, and RTG-2. Firstly, gap junction presence was determined by screening for gap junction protein alpha 7 and alpha 1 (GJA7 and GJA1) presence at the transcript level and GJA7 at the protein level. GJA7 was successfully identified at both the transcript and protein levels, and GJA1 was detected at the transcript level in all three cell lines. This is the first report of a GJA7 full-length transcript sequence in rainbow trout cells. Gap junction function, as determined by gap junction intercellular communication (GJIC), was examined using Lucifer yellow dye migration with the scrape and load technique; visualized by fluorescence microscopy. Phorbol 12-myristate 13-acetate (PMA), a gap junction inhibitor, was used to confirm the presence of functional gap junctions. Effects of serum deprivation on GJIC were also monitored; 24-h serum deprivation resulted in greater dye migration compared with 30-min serum deprivation. Both RTG-2 and RTgill-W1 showed significant dye migration that was inhibited by PMA while RTgutGC did not. Human foreskin fibroblast (HFF-1) cells were used as a positive control for gap junction presence and function. Taken together, our study shows that rainbow trout cells express connexin transcripts and proteins, and RTG-2 and, to a lesser extent, RTgill-W1 cells are able to perform GJIC.

Serum Starvation Induces BACE1 Processing and Secretion.

β-secretase (BACE1) is a type 1 transmembrane protein implicated in Alzheimer's Disease (AD) pathogenesis. Cleavage of Amyloid Precursor Protein (APP), initiated by BACE1 and followed by γ-secretase, leads to the formation of toxic Aβ peptides. Increased levels of BACE1 have been detected in the CSF of AD patients compared to age-matched healthy controls indicating that neurodegenerative conditions induce shedding of BACE1. To mimic such conditions, we examined whether serum deprivation stimulates proteolysis-dependent secretion of BACE1. Detection of BACE1 secretion in BACE1 overexpressing cells or ADAM10/ADAM17 knockout fibroblasts cultured under serum deprivation conditions, using western blot analysis. We found that serum deprivation of U251 neuroblastoma or HEK293T cells overexpressing BACE1 stimulated secretion of BACE1. Using ADAM10/ADAM17 knockout fibroblasts and inhibitors of both ADAM10 and ADAM17, we obtained data indicating that these proteases are involved in serum-starvation induced shedding of BACE1. This is unexpected since BACE1 is localized mainly in lipid rafts while ADAM10 is localized mainly in nonlipid raft domains. We hypothesized that serum deprivation results in alterations in the lipid composition of the membrane which can alter the localization of ADAM10 and BACE1. In support, we obtained results indicating that extraction of membrane cholesterol following incubation with methyl β cyclodextrin potentiated the effect of serum deprivation. Secreted BACE1 was also found to be enzymatically active towards immunoprecipiated full length APP. Serum starvation induces ADAM10-mediated BACE1 secretion.

AealRACK1 expression and localization in response to stress in C6/36 HT mosquito cells.

Insect cells adapt to numerous environmental stressors, including chemicals and invasion of pathogenic microorganisms among others, coordinating cellular and organismal responses. Individual cells sense the environment using receptors that trigger signaling pathways that regulate expression of specific effector proteins and/or cellular responses as movement or secretion. In the coordination of responses to stress, scaffold proteins are pivotal molecules that recruit other proteins forming active complexes. The Receptor for Activated C Kinase 1 (RACK1) is the best studied member of the conserved tryptophan-aspartate (WD) repeat family. RACK1 folds in a seven-bladed β-propeller structure and it could be activated during stress, participating in different signaling pathways. The presence and activities of RACK1 in mosquitoes had not been documented before, in this work the molecule is demonstrated in an Aedes albopictus-derived cell line and its reaction to stress is observed under the effect of serum deprivation and the presence of glucocorticoid analog dexamethasone, a chemical used to cause stress in vitro.

A preliminary study of serum-deprivation-induced release of S100A13 and flbroblast growth factor 1 from thyroid cancer TT cell

Objective To investigate the effect of serum-deprivation on the changes of [ Ca2+ ] i and the protein release of S100A13 and fibroblast growth factor 1 ( FGF-1 ) in thyroid cancer TT cell,and to reveal the role of Ca2+in the protein release of S100Al3 and FGF-1.Methods The protein expressions of FGF-1 and S100A13 in TT cells under serum-deprivation were detected by Western blot.The released FGF-1 protein from TT cells in the supernatant fluid was detected by ELISA.Realtime dynamic examinations on the change of 1 h [ Ca2+ ] i in TT cells under serum-deprivation were detected by confocal laser scanning microscopy.Then,the effect of EGTA( 2.5 mmol/ L),BAPTA-AM (2.5 μmol/L)on distributions of the fluorescence of S100AI 3 and FGF-1 in TT cells under serumdeprivation for6 h were detected by indirect immunofluorescence.Results The expressions of FGF-1 and S100A13 in TT cells after serum-deprivation for4 h and 6 h were reduced( P＜0.05 or P＜0.01 ),but the released FGF-1 protein from TT cells in the supernatant fluid was elevated ( P＜0.05 or P＜0.01).Confocal laser scanning of Ca2+ imaging indicated that [ Ca2+ ] i of serum-deprivation TT cells maintained the relative stabilization within 23 win,but the rapid rise of [ Ca2+ ] i achieved peak value 1.6 μmol/L after 30 min,and remained stable for about 17 win,and thereafter 40 win slowly dropped to a low level From 40 win to 60 win the [ Ca2+ ] i was about 0.3-0.6 μ mol/L.The average [ Ca2+ ] i was higher than that in normal group,EGTA group,and BAPTA-AM group within 1 h.The protein expressions of S100A13 and FGF-1 did not drop obviously in EGTA group and BAPTA-AM group.Conlusion The release of S100A13 and FGF-1 from TT cell under serum-deprivation is possibly related with the change of [ Ca2+ ]i.Both Ca2+-chelating agents EGTA and BA PTA-AM are able to inhibit the rise of [ Ca2+ ] i and release of S100A 13 and FGF-1 from TT cells under serum-deprivation. Key words: Serum-deprivation-induced; S100A1 3 ; Fibroblast growth factor 1 ; [ Ca2+ ] i; Ca2+-chelating agent

Short-Term Serum Deprivation Confers Sensitivity to Taxanes in Platinum-Resistant Human Ovarian Cancer Cells

Based on the evidences showing that serum deprivation provokes apoptosis in a variety of cells, we have investigated the effect of serum deprivation on drug sensitivity. After human ovarian cancer cells were preincubated in 0.5 % serum containing medium for 12 hours, cellular drug sensitivities were determined by colony-forming assay. Serum deprivation treatment resulted in significant increase in paclitaxel sensitivity by factors of mean ± SD, 148.6 ± 28.1 and 10.1 ± 1.0 (n = 3; P < 0.001) fold in platinum-resistant C13 and CP70 cells, respectively. Similarly, serum deprivation induced significant docetaxel sensitivity in these cell lines. However, no enhancement effect of serum deprivation was observed in platinum-sensitive 2008 and A2780 cells. Serum deprivation did not have any effect on the sensitivities to cisplatin, vincristin, and doxorubicin in all of these cells. More than 7-fold increase of apoptotic cells were observed in C13 or CP70 cells when they were treated by serum deprivation followed by paclitaxel compared with the treatment of either serum deprivation or paclitaxel alone. Confocal laser microscopy using rhodamine 123 and flow cytometric analysis with 3,3'-dihexyloxacarbocyanine iodide revealed that serum deprivation decreased mitochondrial membrane potential in C13 or CP70 cells, whereas no change was observed in 2008 and A2780 cells. This indicates that serum deprivation induced depolarization specifically in platinum-resistant cells. Electron microscopy revealed that serum deprivation caused regeneration of mitochondrial matrix structure in C13 or CP70 cells where mitochondria were usually destructed and disappeared. These results indicate that serum deprivation confers taxane hypersensitivity specifically in platinum-resistant cells by recovering their impaired mitochondrial functions. The evidence might be clinically beneficial for the development of new chemotherapeutic technology, particularly for the patients with platinum-resistant ovarian cancer.

Hormetic effects of serum deprivation on androgen regulation of dopamine cell viability

Hormesis is the beneficial response of treatments harmful at high concentrations but at low levels confer resistance, such as ischemic preconditioning and dietary restriction. Androgens have been shown to increase the vulnerability of dopamine neurons via an oxidative stress mechanism. In this study, we examined if serum deprivation alters androgens’ effects on oxidative stress in a dopaminergic cell line (N27 cells). N27 cells were exposed to tert‐butyl‐hydrogen peroxide (500uM) 24hrs prior to androgen exposure in either a serum‐free environment or charcoal‐stripped serum environment. Our results showed decreased neuronal vulnerability to oxidative stress in response to androgens in the serum‐free environment compared to the charcoal‐stripped environment. Indicating that serum deprivation had a hormetic effect on androgen regulation in dopamine neuronal viability. To determine underlying mechanisms, N27 cells in a serum‐free environment were exposed to androgens for varying time periods (0–48hrs) and imaged to determine morphology. Analysis showed increased neurite outgrowth after 2hrs of androgen exposure, indicating increased synaptic plasticity. These results suggest that serum deprivation can confer a hormetic effect on dopamine neuronal viability in response to androgens by increasing synaptic plasticity. Supported by NIH grants F32NS061417‐01 to RLC and AG022550 and AG027956 to MS.

Effects of serum deprivation on the mechanical properties of adherent vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) function plays a key role in regulating the development and progression of vascular lesions. Among the more significant phenomena that occur during the development of these lesions is the phenotypic switching of VSMCs from a contractile to a synthetic state. A better understanding of the concurrent changes to VSMC mechanical properties that occur with phenotypic shifts can help to elucidate the role of VSMC mechanics in the development of vascular diseases. In the current study, the mechanical properties of adherent cultured rat aortic VSMCs were assessed by atomic force microscopy. Serum starvation was used to induce a phenotypic shift in vitro. It was concluded that serum starvation led to a statistically significant increase in apparent elastic modulus after 5 days, as well as a statistically significant decrease in hysteresis after culture for 3 days. If this trend of VSMC mechanical properties changing concurrently with phenotypic shifts were to hold true in vivo, such changes could affect the processes of mechanotransduction and/or arterial mechanical properties, thereby contributing to the progression of vascular disease.

The effect of serum withdrawal on the protein profile of quiescent human dermal fibroblasts in primary cell culture

The effect of serum deprivation on proliferating cells is well known, in contrast its role on primary cell cultures, at confluence, has not been deeply investigated. Therefore, in order to explore the response of quiescent cells to serum deprivation, ubiquitous mesenchymal cells, as normal human dermal fibroblasts, were grown, for 48 h after confluence, in the presence or absence of 10% FBS. Fibroblast behaviour (i.e. cell morphology, cell viability, ROS production and elastin synthesis) was evaluated morphologically and biochemically. Moreover, the protein profile was investigated by 2-DE and differentially expressed proteins were identified by MS. Serum withdrawal caused cell shrinkage but did not significantly modify the total cell number. ROS production, as evaluated by the dihydroethidium (DH2) probe, was increased after serum deprivation, whereas elastin synthesis, measured by a colorimetric method, was markedly reduced in the absence of serum. By proteome analysis, 41 proteins appeared to significantly change their expression, the great majority of protein changes were related to the cytoskeleton, the stress response and the glycolytic pathway. Data indicate that human dermal fibroblasts in primary cell culture can adapt themselves to environmental changes, without significantly altering cell viability, at least after a few days of treatment, even though serum withdrawal represents a stress condition capable to increase ROS production, to influence cell metabolism and to interfere with cell behaviour, favouring the expression of several age-related features.

Effects of Serum Deprivation and Cycloheximide on Cell Cycle of Low and High Passage Porcine Fetal Fibroblasts

Arrest of cells in G0/G1 cell cycle phase is desired for nuclear transfer procedures. Serum starvation and cell cycle inhibitors are different ways to induce synchronization of the cell cycle. This study investigated the effects of serum starvation and cycloheximide (CHX) on the cell cycle of low (5th) and high (15th) passages fetal porcine fibroblasts. Cell cycle phases were determined using fluorescent activated cell sorting. Fifth passage fibroblast cultures had higher (p < 0.05) proportion of cells in G0/G1 only after 72 h of serum starvation (77.60 +/- 0.65) when compared with non-starved cells (71.44 +/- 1.88). Serum starvation for all periods tested induced an increase (p < 0.05) on proportion of cells in G0/G1 on the 15th passage. No significant differences were observed on the 5th passage cultures exposed to CHX, although, on the 15th passage an increase on proportion of cells was observed after all periods of exposure (p < 0.05). These data indicates that high passage cells in vitro are more susceptible to serum starvation and CHX G0/G1 synchronization.

Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells

We characterized the expression and functional properties of the ADP-sensitive P2Y(1) and P2Y(12) nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y(12) receptor relative to P2Y(1) was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y(1) receptor was low, and the P2Y(12) receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y(12) receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y(12) receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y(1) receptor, indicating the inhibitory role of P2Y(1) in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y(1) to P2Y(12) would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation.

GDNF abates serum deprivation-induced tyrosine hydroxylase Ser19 phosphorylation and activity

High dopamine levels can contribute to neuronal dysfunction, impair plasticity and be toxic to neuronal cells in pathological conditions. The synthesis of dopamine is regulated by phosphorylation of the rate-limiting enzyme tyrosine hydroxylase (TH) under physiological conditions, with the phosphorylation of Ser31 and Ser40 directly increasing TH activity. Although a third phosphorylation site, Ser19, does not appear to directly regulate TH activity in physiological conditions, its role in pathological conditions is poorly understood. In this study, we examined the effects of serum deprivation (to mimic loss of retrogradely/anterogradely transported target-derived neurotrophic factors following axonal injury) and glutamate receptor stimulation (to mimic excitotoxicity) on TH phosphorylation and activity in a cell line and in mesencephalic primary culture cells. In addition, we also tested whether glial cell line-derived neurotrophic factor (GDNF) can alter these changes. We demonstrate that serum-deprivation resulted in a sustained increase in Ser19 phosphorylation beginning at 3 h and lasting up to 10 h without any detectable change in Ser31 or Ser40 phosphorylation within this time frame. This increase in Ser19 phosphorylation was associated with enhanced TH activity and was due, in part, to glutamate-receptor-mediated calcium influx and possibly calcium/calmodulin-dependent protein kinase II (CaMKII) activation. Interestingly in this serum-deprivation model, GDNF blocked the increase in Ser19 phosphorylation and TH activity at the 10-h time point following serum deprivation. Furthermore, GDNF also blocked the glutamate-mediated increase in Ser19 phosphorylation in rat primary mesencephalic neuronal cultures. Taken together, these findings suggest that GDNF may reduce dopamine synthesis in pathological conditions.

Cell cycle synchronization of embryonic stem cells: Effect of serum deprivation on the differentiation of embryonic bodies in vitro

Research on stem-cell transplantation has indicated that the success of transplantation largely depends on synchronizing donor cells into the G0/G1 phase. In this study, we investigated the profile of embryonic stem (ES) cell synchronization and its effect on the formation of embryonic bodies (EBs) using cell culture with serum deprivation. The D3 cell line of ES cells was used, and parameters such as cell proliferation and activity, EB formation, and expression of stage-specific embryonic antigen-1 and Oct-4 were investigated. Results showed that the percentage of G0/G1 stage in serum deprivation culture is significantly higher than that in culture with serum supplementation. Synchronized ES cells can reenter the normal cell cycle successfully after serum supply. EBs formed from synchronized ES cells have higher totipotency capability to differentiate into functional neuronal cells than EBs formed from unsynchronized ES cells. Our study provides a method for ES treatment before cell transplantation that possibly helps to decrease the rate of cell death after transplantation.

Effects of Serum Deprivation on Expression of Proteolytic-Related Genes in Chick Myotube Cultures

We previously reported that serum deprivation stimulates myofibrillar proteolysis in chick myotubes. In the present study, we examined the effect of serum deprivation on expression of the proteolytic-related genes (ubiquitin, proteasome, calpains, and cathepsin B) by real-time PCR of cDNA in chick myotubes. Myotubes were incubated with serum-free medium for 24 h. Ubiquitin and proteasome subunits (C1 and C2) and calpains (m-, mu-, and p94/calpain-3) but not cathepsin B mRNA expression were increased by serum deprivation. These results indicate that serum deprivation stimulates ubiquitin-proteasome and calpain proteolytic pathways, resulting in an increase in myofibrillar proteolysis in chick myotubes.

Effect of serum deprivation on constitutive production of granulocyte‐colony stimulating factor and granulocyte macrophage‐colony stimulating factor in lung cancer cells

We previously established 2 lung cancer cell lines, OKa-C-1 and MI-4, which constitutively produce an abundant dose of granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF). Many other cases with G-CSF or GM-CSF producing tumors have been reported up to the present. However, the biological properties of the overproduction of G-CSF and GM-CSF by tumor cells have not been well known. Several reports demonstrated the presence of an autocrine growth loop for G-CSF and GM-CSF in nonhematopoietic tumor cells. We showed that exogenous G-CSF and GM-CSF stimulated cell growth in a dose-dependent manner in OKa-C-1 and MI-4 cells. We could detect the presence of G-CSF and GM-CSF receptors in both cell lines by RT-PCR analysis. We have previously shown that inflammatory cytokines, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta enhance the expression of G-CSF and GM-CSF in the cell lines. However, the factors that regulate constitutive production of G-CSF or GM-CSF by tumor cells are still unknown well. In our study, we first reported that serum deprivation stimulated constitutive production of G-CSF and GM-CSF by lung tumor cells through activation of nuclear factor (NF)-kappaB and p44/42 mitogen-activated protein kinase (MAPK) pathway signaling. We suggest that G-CSF and GM-CSF constitutively produced by tumor cells could grow tumor itself and rescue tumor cells from the cytotoxicity of serum deprivation.

Effects of Serum Deprivation on Myofibrillar Proteolysis in Chick Myotube Cultures

We examined the effects of serum deprivation on myofibrillar proteolysis in chick myotubes. Myotubes were incubated with serum-free medium for 24 hours. N(tau)-methylhistidine release, as an index of myofibrillar proteolysis, as well as protease activities such as calpain, proteasome, and cathepsins (B+L and D) activities were increased by serum deprivation. These results indicate that serum deprivation induces calpain, proteasome, and cathepsins activities, resulting in an increase in myofibrillar proteolysis in chick myotubes.

Chemosensitivity of TEL-AML1 Fusion Transcript Positive Acute Lymphoblastic Leukemia Cells

The TEL-AML1 fusion which results from a cryptic t(12;21) translocation is the most frequently occurring genetic abnormality in childhood acute lymphoblastic leukemia (ALL) and has been associated with an excellent treatment outcome. In the present study, we examined the FAS/BCL-2 expression profiles and chemosensitivity of primary leukemic cells from children with newly diagnosed t(12;21)TEL-AML1 fusion transcript-positive versus t(12;21)TEL-AML1 fusion transcript-negative standard risk ALL. TEL-AML1+ ALL cells expressed higher levels of the pro-apoptotic protein Fas and lower levels of the anti-apoptotic protein Bcl2 than TEL-AML1− ALL cells, as determined by confocal laser scanning microscopy. TEL-AML1+ ALL cells were more sensitive to the apoptosis-inducing effects of serum deprivation, dexamethasone and vincristine than TEL-AML1− ALL cells. This study provides novel mechanistic insights regarding the chemosensitivity of TEL-AML1+ ALL cells and provides a cogent explanation for the excellent leukemia-free survival outcome of children with TEL-AML1+ ALL treated on contemporary chemotherapy programs.

Effects of serum deprivation, insulin and dexamethasone on polysome percentages in C2C12 myoblasts and differentiating myoblasts

An increase in the rate of protein synthesis in living cells can be achieved by regulating the quantity of mRNA, ribosomes, and enzymes available for translation or by regulating the efficiency at which existing components are used. Efficiency can be measured by comparing the number of ribosomes actively engaged in the synthesis of protein (polysomes) to the pool of free ribosomes. The objective of this study was to determine the percentage of ribosomes found as polysomes in C2C12 cells deprived of serum or exposed to insulin or dexamethasone 24 h before and after being stimulated to differentiate. Individual 60 mm culture dishes were exposed to serum-free control medium, medium containing serum, insulin, or dexamethasone for a period of 1 h or 2 h and then quickly frozen. The ribosomes and polysomes from these cells were separated by ultracentrifugation on 15 to 60% sucrose gradients and the absorbance across the gradient at 254 nm was recorded. Polysome percentages were determined as the area under the polysome peak divided by the total area under the curve. Serum deprivation caused a 12% decline in the percentage of ribosomes found as polysomes (P< 0.01). Dexamethasone caused a quadratic decline (P< 0.05) in polysome percentage, while insulin yielded a quadratic increase (P< 0.05). Protein synthesis assays measuring3H-tyrosine uptake showed similar responses. These changes occurred in the absence of any differences in total RNA concentration. It was concluded that differentiation and the absence of serum in the media reduced the rate of recruitment of ribosomes for protein synthesis. Insulin increased ribosome recruitment which was also observed by a similar increase in incorporation of radio-labeled tyrosine.