Development and future prospects of selective organometallic compounds as anticancer drug candidates exhibiting novel modes of action.

Organometallic complexes have widely been used for the treatment of various diseases viz., malaria, arthritis, syphilis, pernicious anemia, tuberculosis and particular in cancers. Recent decades have witnessed an upsurging interest in the application of organometallic compounds to treat various phenotypes of cancers with multiple etiologies. The unique and exceptional properties of organometallic compounds, intermediate between classical inorganic and organic materials provide new insight in the progress of inorganic medicinal chemistry. Herein, we have selectively focused on various organometallic sandwich and half-sandwich complexes of ruthenium (Ru), titanium (Ti), gold (Au) and iron (Fe) exhibiting promising activity towards a panel of cancer cell lines and resistant cancer cell lines. These complexes exhibit novel mechanisms of drug action through incorporation of outer-sphere recognition of molecular targets and controlled activation features based on ligand substitution along with monometallic and heterometallic redox processes. Furthermore, they are usually found to be uncharged or neutral possessing metals in a low oxidation state, exhibit kinetic stability, relative lipophilicity and are amenable to a host of various chemical transformations. This review mainly sheds light on the successful advancement of organometallic complexes as anticancer drug aspirants in relation to their versatile structural chemistry and innovative mechanisms of action targeting nucleic acids, several enzymes viz; thioredoxin reductases (Thrx), EGFR, transferrin, cathepsin B, topoisomerases etc.

Comparison of the effects of nimodipine and deferoxamine on brain injury in rat with subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) may lead to brain atrophy and cognitive dysfunction. This study aimed to compare the efficacy of nimodipine and deferoxamine on these sequelae of SAH. A rat model of SAH was established by the double-hemorrhage method. These rats were injected with saline (intraperitoneal, IP), nimodipine (IP), or deferoxamine (IP and intranasal) every 12 h for 5 days after SAH. The MRI scanning, including magnetic resonance angiography, diffusion tensor imaging, T2-weighted imaging, was performed to detect the brain structure. The levels of iron metabolism-related proteins were examined by Western blot analysis. The Morris water maze (MWM) test was used to assess the cognitive function. Then, then neurons in the cortex and hippocampus were counted on hematoxylin and eosin-stained brain sections. Significant cerebral vasospasm (CVS) was found in the saline and deferoxamine groups, but not in the nimodipine group. Cerebral peduncle injury was detected in the saline and nimodipine groups, but not significantly in the deferoxamine group. Compared with nimodipine, deferoxamine reduced transferrin (Tf), Tf receptor, and ferritin levels after SAH. The MWM performances were significantly worse in the saline and nimodipine groups than that in the deferoxamine group. Brain atrophy and neuronal losses were more significant in the saline and nimodipine groups than in the deferoxamine group. Nimodipine significantly ameliorated CVS, but it did not improve the late changes in brain structure and cognitive function. Deferoxamine effectively reduced neuronal cell death and ameliorated cognitive function after SAH.

Characterization of the interaction between carbon black and three important antioxidant proteins using multi spectroscopy and modeling simulations.

A major user of carbon black is the pigment and dyes industry, where carbon black is incorporated into paints, inks, printers, and plastics. However, little is known about the mechanism underlying the toxicity of carbon black to antioxidant proteins. Carbon black can cause oxidative stress to organisms after they invade into the body. Antioxidant proteins play a key role in keeping the organism from nanoparticle-induced oxidative damage and tend to bind with nanoparticles immediately after their invading into the biological environment, so it is meaningful to elucidate the toxicity of nanoparticles on the antioxidant proteins. In this study, the toxicity of carbon black (SB100) on three different antioxidant proteins (TF (transferrin), SOD (superoxide dismutase), and LYZ (lysozyme)) were investigated. The multi-spectra studies indicated that SB100 interacted with these three proteins and changed their structure in different ways. SB100 changed the microenvironment of fluorophores in SOD and LYZ by quenching the fluorescence spectra of the two enzymes, while changed that of TF by increasing the fluorescence intensity of TF. SB100 changed the secondary structure of these three proteins by decreasing the α-helix content of TF and increasing that of SOD and LYZ. Moreover, SB100 changed the hydrophobicity of the three proteins in different ways as well. And SOD exhibits a more severe activity inhibition than LYZ after exposed to SB100. In summary, SB100 caused different structural and functional changes to these three antioxidant enzymes.

Rapid chemical de-N-glycosylation and derivatization for liquid chromatography of immunoglobulin N-linked glycans.

Glycan analysis may result in exploitation of glycan biomarkers and evaluation of heterogeneity of glycosylation of biopharmaceuticals. For N-linked glycan analysis, we investigated alkaline hydrolysis of the asparagine glycosyl carboxamide of glycoproteins as a deglycosylation reaction. By adding hydroxylamine into alkaline de-N-glycosylation, we suppressed the degradation of released glycans and obtained a mixture of oximes, free glycans, and glycosylamines. The reaction was completed within 1 h, and the mixture containing oximes was easily tagged with 2-aminobenzamide by reductive amination. Here, we demonstrated N-linked glycan analysis using this method for a monoclonal antibody, and examined whether this method could liberate glycans without degradation from apo-transferrin containing NeuAc and NeuGc and horseradish peroxidase containing Fuc α1-3 GlcNAc at the reducing end. Furthermore, we compared glycan recoveries between conventional enzymatic glycan release and this method. Increasing the reaction temperature and reaction duration led to degradation, whereas decreasing these parameters resulted in lower release. Considering this balance, we proposed to carry out the reaction at 80°C for 1 h for asialo glycoproteins from mammals and at 50°C for 1 h for sialoglycoproteins.

In Vitro Release Kinetics and Transferrin Saturation Study of Intravenous Iron Sucrose Entrapped in Poly(ethylene glycol)-Assisted Silica Xerogel.

The presence of labile iron fractions in intravenous iron supplements compromises their safety. Poly(ethylene glycol) (PEG)-assisted silica xerogel was evaluated as a potential drug carrier for iron sucrose with the purpose of limiting labile iron available for in vitro uptake by transferrin. The drug entrapped xerogels were synthesized by the sol-gel process with varying amounts of PEG. In vitro release studies were conducted in simulated body fluid (SBF) at 37 ± 0.02 °C (pH 7.4). The results indicated that the cumulative release percentage increased with the increase in the amount of PEG in the matrix. The biphasic release profile followed first-order kinetics for the first 6 h and Higuchi model for the remaining time (up to 168 h). The sample showing highest percentage of cumulative release (the xerogel with 16 % PEG) was used for in vitro transferrin saturation studies in contrast with the plain drug. The xerogel formulation exhibited 7.25 ± 0.4 % transferrin saturation in 180 min as compared to 12.89 ± 0.2 % for the raw drug. These results indicate that encapsulation of iron sucrose in PEG-assisted silica xerogel and subsequent sustained release from the matrix can improve the safety of the drug when presence of labile iron is a major concern.

Transferrin receptors and glioblastoma multiforme: Current findings and potential for treatment.

The current standard treatment for glioblastoma multiforme (GBM) is surgery followed by chemotherapy and external radiation. Even with the standard treatment, the 2 year survival rate for GBM is less than 20%, making research for alternative treatments necessary. Transferrin receptor 1 (TfR1) controls the rate of cellular iron uptake by tuning the amount of iron delivered to the cells to meet metabolic needs. Kawabata et al. (J Biol Chem 1999;274:20826-32) cloned a second TfR molecule known as transferrin receptor 2 (TfR2) in 1999. Multiple experimental studies have documented increased expression of TfR1 on both proliferating cells and cells that have undergone malignant transformation. Calzolari et al. concluded that TfR2 is frequently expressed in human cell lines in 2007 (Blood Cells Mol Dis 2007;39:82-91) and in GBM in particular in 2010 (Transl Oncol 2010;3:123-34). In GBM, a highly significant correlation (p<0.0001) was found between the expression level of TfR2 and overall survival, showing that higher levels of TfR2 expression were associated with an overall longer survival. The data on which of the two transferrin receptors is the better target is also unclear and should be studied. The transferrin pathway may be a promising target, but more research should be completed on the antigenicity to discern the viability of it as an immunotherapy target.

Does combined peroxisome proliferator-activated receptors-agonist and pravastatin therapy attenuate the onset of diabetes-induced experimental nephropathy?

OBJECTIVES: To investigate the combined effects of rosiglitazone and pravastatin on renal functions in early streptozotocin induced diabetic nephropathy (DN). METHODS: This study was carried out at King Khalid University Hospital Animal House, Riyadh, Saudi Arabia from August 2013 to February 2014. Fifty male Wistar rats were assigned to normal control rats and diabetic rats that received saline, rosiglitazone, pravastatin, or rosiglitazone+pravastatin for 2 months. Their weight range was 230-250 gm, and age range was from 18-20 weeks. At the end of experiment, creatinine clearance, and urinary albumin to creatinine ratio (ACR) were measured. Blood samples were analyzed for transferrin, glycosylated hemoglobin (HbA1c), lipid profile, tumor necrosis factor-alpha (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and lipid peroxide. RESULTS: Rosiglitazone treatment increased creatinine clearance and plasma transferrin, and decreased urinary ACR, HbA1c, plasma TNF-α, ICAM-1, and serum lipid peroxide levels without affecting the altered lipid profile. Pravastatin treatment produced similar results and normalized the lipid alteration. The combination of rosiglitazone and pravastatin was more effective in attenuating the diabetes-induced nephropathy compared with treatment with either drug alone. CONCLUSION: The combination strategy of rosiglitazone and pravastatin may provide a potential synergistic renoprotective effect against DN by improving renal functions and reducing indices of DN. 

False negativity to carbohydrate-deficient transferrin and drugs: a clinical case.

INTRODUCTION: In this work we report on the possible effect of the medical therapy on CDT concentration in a chronic alcohol abuser, with known medical history (July 2007 - April 2012) and alcohol abuse confirmed by relatives. CASE HISTORY: At the end of 2007, patient displayed the following laboratory results: AST 137 U/L, ALT 120 U/L, GGT 434 U/L, MCV 101 fL and CDT 3.3%. On December 2007, after double coronary artery bypass surgery, he began a pharmacological treatment with amlodipine, perindopril, atorvastatin, isosorbide mononitrate, carvedilol, ticlopidine and pantoprazole. In the next months, until may 2011, the patient resumed alcohol abuse, as confirmed by relatives; however, CDT values were repeatedly found negative (0.8% and 1.1%) despite elevated transaminases and GGT, concurrent elevated ethyl glucuronide concentration (> 50 mg/L) and blood alcohol concentration (> 1 g/L). Alcohol consumption still continued despite increasing disulfiram doses ordered by an Alcohol Rehab Center. On May 2011, the patient was transferred to a private medical center where he currently lives. CONCLUSIONS: This study suggests the possibility that a medical therapy including different drugs may hamper the identification of chronic alcohol abusers by CDT.

New Ga derivatives of the H2dedpa scaffold with improved clearance and persistent heart uptake.

Recent advances in positron emission tomography (PET)/computed tomography have fueled the development of new PET-isotope-based agents for myocardial perfusion imaging. (68)Ga, a generator-produced PET isotope, is an attractive radionuclide for developing a (68)Ga-based cardiac imaging agent. We have synthesized seven new chelate systems based on our previously reported 1,2-[{6-(carboxylato-)pyridin-2-yl}methylamino]ethane (H(2)dedpa) scaffold. These ligands form lipophilic, cationic complexes upon coordination of (67/68)Ga(III) under mild, direct labeling conditions within 10 min at room temperature. The corresponding cold complexes were also synthesized, and the solid-state structure of one of the complexes, [Ga(19)][ClO(4)], was determined. All compounds were investigated for in vitro stability against transferrin, and log P values were determined. In vivo biodistribution studies in mice showed that four of the seven investigated complexes provided greatly improved blood, lung and kidney clearance compared to previously reported derivatives. Two complexes with log P>1.1 exhibited persistent heart uptake over the course of 2 h above 1% ID/g.

1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation.

The α-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB's cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37°C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other α-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml-1) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(•), and those with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(•) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 µM) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity.

Effect of intradialytic intravenous administration of omega-3 fatty acids on nutritional status and inflammatory response in hemodialysis patients: a pilot study.

OBJECTIVE: Because omega-3 polyunsaturated fatty acids (PUFAs) may have anti-inflammatory properties, we tested the hypothesis that intradialytic, intravenous omega-3 PUFA treatment, combined with dietary supplementation, can modify the inflammatory response to dialysis, and influence the nutritional status of hemodialysis (HD) patients. METHODS: Twenty HD patients with serum albumin at <39g/L received 100mL of 10% omega-3 PUFA emulsion during 11 consecutive HD sessions. Body mass index (BMI), serum albumin, transferrin, and lipids were measured before and after treatment. Serum interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hsCRP) levels were determined before and after the HD session at baseline and after 4 weeks of treatment. RESULTS: No adverse events were evident during the study. There were no significant changes in BMI, serum albumin, transferin, total and low-density lipoprotein cholesterol, and triglycerides. Predialysis hsCRP and IL-6 did not change. There was a significant increase in hsCRP (P=.01) and a tendency of IL-6 concentration to increase during the HD session before treatment (P=.067). In contrast, neither hsCRP (P=.21) nor IL-6 (P=.26) changed during the final HD session. Neither urea reduction ratio nor Kt/V changed significantly during the study, but the normalized protein catabolic ratio increased after treatment (P=.003). CONCLUSIONS: Short-term parenteral administration of omega-3 PUFA is safe and well-tolerated by HD patients. The intervention does not significantly influence markers of inflammation or change the nutritional status of chronic HD patients, but it may attenuate the inflammatory response to HD sessions.

Intravenous iron versus erythropoiesis-stimulating agents: friends or foes in treating chronic kidney disease anemia?

Patients with chronic kidney disease (CKD), especially those requiring maintenance hemodialysis treatments, may lose up to 3 g of iron each year because of frequent blood losses. Higher doses of erythropoiesis-stimulating agents (ESAs) may worsen iron depletion and lead to an increased platelet count (thrombocytosis), ESA hyporesponsiveness, and hemoglobin variability. Hence, ESA therapy requires concurrent iron supplementation. Traditional iron markers such as serum ferritin and transferrin saturation ratio (TSAT) (ie, serum iron divided by total iron-binding capacity [TIBC]), may be confounded by non-iron-related conditions. Whereas serum ferritin <200 ng/mL suggests iron deficiency in CKD patients, ferritin levels between 200 and 1,200 ng/mL may be related to inflammation, latent infections, malignancies, or liver disease. Protein-energy wasting may lower TIBC, leading to a TSAT within the normal range, even when iron deficiency is present. Iron and anemia indices have different mortality predictabilities, in that high serum ferritin but low iron, TIBC, and TSAT levels are associated with increased mortality, whereas hemoglobin exhibits a U-shaped risk for death. The increased mortality associated with targeting hemoglobin above 13 g/dL may result from iron depletion-associated thrombocytosis. Intravenous (IV) iron administration may not only decrease hemoglobin variability and ESA hyporesponsiveness, it may also reduce the greater mortality associated with the much higher ESA doses that have been used in some patients when targeting higher hemoglobin levels.

Intravenous iron in chronic kidney disease: haemoglobin change shortly after treatment of patients neither on dialysis nor on erythropoietin.

Anaemia is a common in chronic kidney disease. Although erythropoietin and iron supplementation are established treatments, knowledge on the use of IV iron alone in patients not on dialysis or erythropoietin is incomplete. The responses of 82 patients referred to the renal anaemia service with haemoglobin of 11.5 g/dl or less were assessed 1 week after completing four once weekly doses of 200 mg of venofer. No patients were on dialysis or erythropoietin. The haemoglobin rise 1 week after treatment was 0.53 g/dl. Ferritin levels improved from 110.8 to 410.2 ng/l and transferrin saturation from 17.7 to 27.3%. Ferritin levels remained below our target range (200-500 ng/l) in 7.7% while 25.6% had levels above this. Ferritin levels remained less than 800 ng/l in nearly all patients. Intravenous iron is cost effective and should be considered for use in patients with renal anaemia. Patients with CKD stage 5 appeared to respond less well.

HPLC and mass spectrometric characterization of a candidate reference material for the alcohol biomarker carbohydrate-deficient transferrin (CDT).

BACKGROUND: Measurement of the alcohol-induced change of the serum transferrin glycoform pattern, carbohydrate-deficient transferrin (CDT), is used as a biomarker for heavy drinking. This study characterized a candidate reference material for CDT measurement derived from isolated human transferrin glycoforms. METHODS: Four transferrin glycoforms were separated from human plasma by standard methods. The identity and purity of the fractions was evaluated by HPLC, using specific absorbance measurement of the iron-transferrin complex at 470 nm, and by mass spectrometry, using ESI Q-Tof MS. A primary candidate reference material was prepared by mixing isolated fractions in transferrin-free plasma in a proportion similar to that in serum and with 0-12% disialotransferrin. A secondary candidate reference material was prepared by spiking a serum pool with 1-9% disialotransferrin. RESULTS: Initial identification of the isolated transferrin fractions as disialo-, trisialo-, tetrasialo- and pentasialotransferrin was based on agreement with established HPLC retention times for authentic serum samples (RRT 0.998-1.004). The presence of single symmetric peaks suggested that the fractions were sufficiently pure. The identity and purity was further based on MS agreement of observed with theoretical molecular masses (Delta(m)<0.03%). The %disialotransferrin target values for the secondary candidate reference material showed good correlation with the measured results by an HPLC candidate reference method (r(2)=0.999). CONCLUSIONS: The separated human transferrin fractions used to prepare the CDT candidate reference material were indicated to contain distinct glycoforms. Having access to a CDT reference material in serum matrix will facilitate comparison of results between different methods and aid in the standardization process.

Silybin treatment is associated with reduction in serum ferritin in patients with chronic hepatitis C.

GOALS: The goal of this study was to examine the effect of a standardized silybin and soy phosphatidylcholine complex (IdB 1016) on serum markers of iron status. BACKGROUND: Milk thistle and its components are widely used as an alternative therapy for liver disease because of purported antioxidant, anti-inflammatory, and iron chelating properties. STUDY: Thirty-seven patients with chronic hepatitis C and Batts-Ludwig fibrosis stage II, III, or IV were randomized to 1 of 3 doses of IdB 1016 for 12 weeks. Serum ferritin, serum iron, total iron binding capacity, and transferrin-iron saturation were measured at baseline, during treatment, and 4 weeks thereafter. Wilcoxon signed rank tests were used to compare baseline and posttreatment values. RESULTS: There was a significant decrease in serum ferritin from baseline to end of treatment (mean, 244 vs. 215 mug/L; median, 178 vs. 148 mug/L; P=0.0005); 78% of subjects had a decrease in serum ferritin level. There was no significant change in serum iron or transferrin-iron saturation. Multivariate logistic regression analysis in a model that included dose, age, sex, HFE genotype, history of alcohol use, and elevated baseline ferritin levels demonstrated that stage III or IV fibrosis was independently associated with decreased posttreatment serum ferritin level. CONCLUSIONS: Treatment with IdB 1016 is associated with reduced body iron stores, especially among patients with advanced fibrosis stage.

In vivo study of propolis supplementation effects on antioxidative status and red blood cells.

In vivo study has been conducted on 47 healthy women and men in order to investigate whether daily intake of powdered propolis extract during 30 days has any influence on the following blood parameters: activity of superoxide dismutase, glutathione peroxidase and catalase, concentration of plasma malondialdehyde, total cholesterol, low- and high-density lipoprotein cholesterol, triglycerides, glucose, uric acid, ferritin and transferrin, together with routine red blood cell parameters. The effect of daily propolis intake seems to be time and gender related. For the men test group after the initial 15 days of propolis treatment, 23.2% (p=0.005) decrease in concentration of malondialdehyde was observed. After 30 days of treatment, statistically significant (p=0.010) 20.9% increase in superoxide dismutase activity and change in some of the red blood cell parameters were detected. For the women test group, the propolis treatment did not induce a change in any of the measured parameters.

[The influence of the bioflanoid dicvertin on the antioxidative system ceruloplasmin-transferrin and lipid peroxidation in patients suffering from stable coronary heart disease with dyslipidemia].

Thirty-two patients with coronary heart disease (CHD)--exertional angina and postinfarction cardiosclerosis with dislipidemia--were treated with dicvertin (2,3-dihydro-3,5,7-trihydroxi-2(3,4-dihydroxiphenyl)-4H-1-benzopyran-4-on), a Russian antioxidant, during two months in a day dose of 80 mg in addition to conventional cardial therapy. The therapy resulted in positive changes including improvement of CHD clinical picture and biochemical serum indices, such as a 6% decrease in cholesterol level, a 12% decrease in low-density lipoproteins cholesterol level, and a 14% increase in high-density lipoproteins cholesterol level. The intensity of lipid peroxidation decreased, the levels of diene conjugates and TBA-reactive products lowered by 38% and 40%, respectively. The fibrinogen level lowered by 20%. The antioxidative status of the patients increased.

[Serum iron parameters in chronic hepatitis C patients and comparison of the results before and during antiviral treatment]. / Ocena stezenia zelaza, ferrytyny, calkowitej zdolnosci wiazania zelaza oraz wysycenia transferyny w surowicy chorych na przewlekle zapalenie watroby typu C oraz analiza wplywu leczenia przeciwwirusowego na wymienione parametry.

OBJECTIVE: The aim of the study was assess serum iron parameters in patients with chronic hepatitis C (CHC) in comparison with control group and evaluate the influence of antiviral treatment on these results. MATERIAL AND METHODS: Adults with confirmed CHC (n=84) and 30 healthy individuals, were included. In all serum iron and ferritin concentration, transferrin saturation (TSAT) and total iron binding capacity (TIBC) were examined. In 25 cases treated with IFN ribavirin tests were repeated between 6-8 month of the therapy. RESULTS: We found significant differences in ferritin concentrations in CHC patients. The mean serum iron concentration was higher in patients above 40 years old (p<0.05). In the cases with ALT activity above 2.5 times the normal range we found higher ferritin concentration (p<0.05). An increase in the serum markers of iron metabolism was more frequently found in subjects with fibrosis (S3-S4) but the differences among groups were not statistically significant. The results of serum iron parameters during antiviral treatment were lower than before therapy but statistical significance was found only in ferritin concentration in woman. CONCLUSIONS: Elevated serum iron, ferritin levels and iron transferrin saturations in subjects with chronic hepatitis C has been noted nearly in a half of examined patients. Statistically significant differences were found in iron and ferritin concentrations. Antiviral treatment did not radically change the serum markers of iron metabolism.

Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells.

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.

Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature.

The effects of bafilomycin, nocodazole, and reduced temperature on recycling and the lysosomal pathway have been investigated in various cultured cell lines and have been shown to vary dependent on the cell type examined. However, the way in which these treatments affect recycling and transport to lysosomes within the same cell line has not been analyzed. In the current study, we used fluorophore-labeled transferrin and dextran as typical markers for the recycling and the lysosomal pathways, respectively, to explore the morphology and the intravesicular pH of endocytic compartments in HeLa cells. The V-ATPase inhibitor bafilomycin selectively inhibited the transport of marker destined for lysosomal degradation in early endosomes, whereas the transport of transferrin to the perinuclear recycling compartment (PNRC) still occurred. The kinetics of transferrin acidification was found to be biphasic, indicative of fast and slow recycling pathways via early endosomes (pH 6.0) and PNRC (pH 5.6), respectively. Furthermore, the disruption of microtubules by nocodazole blocked the transport of transferrin to the PNRC in early endosomes and of lysosome-directed marker into endosomal carrier vesicles. In contrast, incubation at 20 degrees C affected the lysosomal pathway by causing retention of internalized dextran in late endosomes and a delay in transferrin recycling. Taken together, these data clearly demonstrate, for the first time, that the transferrin recycling pathway and transport of endocytosed material to lysosomes are differentially affected by bafilomycin, nocodazole, and low temperature in HeLa cells. Consequently, these treatments can be applied to investigate whether internalized macromolecules such as viruses follow a recycling or degradative pathway.