Protein profiles of the Chinese hamster ovary cells in the resting and proliferating stages

Abstract

Identification and characterization of the proteins that regulate the transition from the resting stage (G0) through G1 to S phase of the cell cycle are of central importance to understand the control of cell proliferation and chromosome replication. Unlike in lower organisms, where relatively small numbers of key factors are involved in this process, the factors involved in the same control mechanisms in mammalian systems are much more complex. Furthermore, accumulating lines of evidence now suggest that the nuclear matrix and chromatin organization also play an essential role for the cell cycle control in mammalian cells. To gain a better understanding of the overall dynamics and changes of the protein factors in the context of matrix/chromatin organization, we examined the protein profiles of the Chinese hamster ovary (CHO) cells in different cell cycle compartments. The methods used in this study included subcellular fractionations (cytosol, nuclear extraction, chromatin, and nuclear matrix), two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), silver staining, and immunoblotting. As expected, significant changes of protein profiles were observed when cells entered into proliferating stages from G0. Among approximately 1200 protein spots analyzed by 2-D PAGE, at least 12 showed marked increase or decrease at this transitional period. Further cell-cycle progression from G1 to S phase showed less dramatic changes of overall protein protile. However, the profile of certain proteins showed rather dramatic changes of their subcellular localization during this transitional period. In particular, the levels of proliferating cell nuclear antigen (PCNA) in the nuclear matrix and chromatin dramatically increased in mid-G1 and in the beginning of S phase, respectively, while the overall PCNA level was relatively constant throughout the cell cycle.

The Post-translational Modifications of Proliferating Cell Nuclear Antigen

“…The reaction was stopped with 10% acetic acid solution. Finally, the gel was dried in the heat oven after it was placed between two sheets of cellophane (Bio-Rad) fastened in two frames as described previously (15).…”

“…The suppliers of other reagents are as follows: protease inhibitor mixture, Roche Diagnostics; bovine histone deacetylase, Calbiochem; IPG gel strips, IPG buffers, DryStrip cover fluid, polyvinylidene difluoride (PVDF) membrane, solution for the ECL reaction, and poly(dA)-(dT) [12][13][14][15][16][17][18] , Amersham Biosciences; dialyzed fetal bovine serum, Invitrogen; anti-PCNA monoclonal antibody PC10, NeoMarkers (Fremont, CA); anti-PCNA-AC monoclonal antibodies (agarose-conjugated), polyclonal anti-HDAC1, anti-DNA polymerases ␤ and ␦ antibodies, peroxidase-conjugated secondary anti-goat antibodies, protein A/G-agarose beads, and normal mouse IgG, Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal anti-acetyl protein antibody, Novus Biologicals (Littleton, CO); peroxidase-conjugated secondary anti-rabbit antibodies, Pierce; Cell-cycle Synchronization-The arrests of cell cycle in G 0 and at the G 1 /S boundary were achieved by isoleucine deprivation and by the combination of isoleucine starvation and mimosine treatment, respectively (15,16).…”

“…Subcellular Fractionation-Protein isolation and subcellular fractionation were as described previously (15,17,18). One notable modification was the use of digitonin in the preparation of cytosol fraction.…”

“…Two-dimensional Gel Electrophoresis-The procedure was as described previously (8,15). Briefly, samples (up to 2 mg of protein) were typically solubilized in lysis buffer (LB) (9 M urea, 4% CHAPS, 1% DTT, 2% IPG buffer, pH 3-10, protease inhibitors mixture, 0.1 mM MG132 proteosome inhibitor, 0.001% bromphenol blue).…”

Proteomic profiling of recombinant cells from large-scale mammalian cell culture processes

“…Additionally electrospray ionisation (ESI-MS/MS) techniques are capable of providing amino acid sequence information on peptide fragments of the parent protein (Mann et al 2001). A number of 2D electrophoresis maps containing the location of identified proteins have been generated for CHO cells (Naryzhny and Lee 2001;Hayduk et al 2004;Champion et al 1999) and NS0 cells (Smales et al 2004). …”