Volume 2 • Issue 1 • 1000105 Altern Integ Med ISSN: AIM, an open access journal Open Access Research Article Alternative and Integrative Medicine Spera et al., Altern Integ Med 2013, 2:1 http://dx.doi.org/10.4172/aim.1000105 Keywords: 1 H–NMR; Mass spectrometry; CHO–K1; cAMP; Cell cycle Abbreviation Magnetic Resonance; MS=Mass Spectrometry; cAMP=Adenosine 3’,5’ Cyclic Monophosphate; RT= Reverse Transformation; Myh10=myosin, Heavy Chain 10, Non-Muscle; Gna11=Guanine Nucleotide Binding Protein, Alpha 11; S61A1=Sec61 Alpha 1 Subunit; CD82=Cluster of Diferentiation 82; FCS=Fetal Calf Serum; TSP=Trimethylsilyl Propionate; ppm=Part Per Million; HPLC=High Performance Liquid Chromatography; MALDI TOF=Matrix Assisted Laser Desorption Ionization Time of Flight; HCCA=α-Cyano-4-Hydroxycinnamic Acid; TFA=Trifuoroacetic Acid; PMSF=Phenylmethanesulfonyl Fluoride; DTT= Dithiothreitol; rpm=Revolutions per minute; G6PD=Glucose-6-Phosphate Dehydrogenase; NADP+=Nicotinamide Adenine Dinucleotide Phosphate; NAD+=Nicotinamide Adenine Dinucleotide; LDH= Lactate Dehydrogenase; S.D.=Standard Deviation; nmMHC=Nonmuscle Myosin Heavy Chain; nmMHC-A=Nonmuscle Myosin Heavy Chain A; nmMHC-B=Nonmuscle Myosin Heavy Chain B; ATP=Adenosine 5’-Triposphate. Introduction Chinese hamster ovary fbroblasts (CHO–K1) cells are a good model to study the transformation of microtubules and nuclear DNA during cell cycle. Te standard CHO-K1 cell displays the classical stigmata of malignantly transformed cells grown in vitro [1]; it has been reported that adenosine 3’,5’ cyclic monophosphate (cAMP) causes cell diferentiation [2] and in particular it causes CHO-K1 cells to lose these transformation characteristics and to assume the morphological habitus approaching that of normal fbroblasts. Tis process is named “reverse transformation” (RT) [3]. It was found that untreated and cAMP treated cells difer only in terms of the three-dimensional organisation and orientation of microtubules and are quite similar in terms of the amount of polymerised tubulin [3-7]. Microtubules and chromatin-DNA structure have impact on various biological processes such as the control of gene expression, the protein synthesis, the cell cycle regulation and the cell transformation [8-11]. Transformed cells frequently exhibit instability in chromosome number even in clonal populations. Defects in the cytoskeletal structure could well be refected in corresponding defects in the spindle, which is made of microtubules and which would result in errors of chromosomal distribution among the cell progeny. However, the changes in the concentration of the diferent metabolites and the diferent protein composition associated with morphological changes observed are largely unknown [4]. Puck et al. proposed that the cytoskeleton transmits information from the cell membrane to chromosomal loci in the nucleus and is an important element in regulation of the exposure process [12,13]. Te mechanism of the transformation in properties here described is as yet obscure. Te phenomena make clear that a fundamental change in properties of the cell membrane has occurred. We had analyzed the CHO-K1 cell protein composition, in particular of the nuclear compartment, by mass spectrometry (MS) to identify changing in the protein pattern before and afer the reverse transformation [14]. We identifed three proteins, Myh10, Gna11 and S61A1 present in the nucleus of untreated cells and absent afer cAMP exposure. Moreover we identifed another protein, Cd82Mouse, which concentration is halved afer the diferentiation. We choose 1 H-NMR since it is revealed an extremely useful and a noninvasive technique in monitoring cell metabolism [15–18]. Since it is known that CHO–K1 cells show diferent growth features depending on the growth medium, we frst studied the variation in CHO–K1 metabolism with the addition of 10% fetal calf serum (FCS), observing *Corresponding author: Claudio Nicolini, President Nanoworld Institute Fondazione EL.B.A.Nicolini, Largo Redaelli 7, Pradalunga, Bergamo, Italy, E-mail: claudio.nicolini@unige.it Received February 03, 2013; Accepted March 04, 2013; Published March 07, 2013 Citation: Spera R, Vasile F, Pechkova E, Nicolini C (2013) Correlation of Changes of Cho–K1 Cells Metabolism to Changes in Protein Expression in Camp Differentiation. Altern Integ Med 2: 105. doi:10.4172/aim.1000105 Copyright: © 2013 Spera R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Correlation of Changes of Cho–K1 Cells Metabolism to Changes in Protein Expression in Camp Differentiation Spera R, Vasile F, Pechkova E and Nicolini C* Nanoworld Institute , Fondazione EL.B.A.Nicolini, Pradalunga, Bergamo, and Laboratory of Nanobiotechnology and Biophysics, Genoa University, Genova, Italy Abstract A correlated investigation of cell metabolism and protein expression of Chinese Hamster Ovary cells (CHO-K1) under different growth conditions was performed by 1 H nuclear magnetic resonance ( 1 H-NMR), mass spectrometry (MS) and biochemical assays. CHO fbroblasts have shown different metabolic products when grown in different media and when differentiation is stimulated by adenosine 3’, 5’ cyclic monophosphate (cAMP). In particular, while addition of Fetal Calf Serum causes an increase in the glucose metabolism correlated to changes in lipid composition of the membrane observed by 1 H-NMR, differentiation induced by cAMP causes biochemical differences in glucose and lipidic metabolism uniquely correlated both to the specifc changes in the composition of nuclear proteins, revealed by mass spectrometry, and to the differences in metabolism, determined by NMR. CHO-K1=Chinese Hamster Ovary cells; 1 H-NMR= 1 H Nuclear Te goal of this paper is to investigate the correlations between changing in the cellular metabolism and in the cellular protein expression afer RT, in order to obtain more information on this cellular process. To this aim we coupled two powerful techniques: nuclear magnetic resonance ( 1 H-NMR) and MS. We think that this approach could be of wide application in the study of many cellular systems.