Biomedical subjects

Pinghu Liu

Publications and source records attributed to Pinghu Liu.

4 recordsLinked to original sources

PubMed2004

Wild-type p16INK4a suppresses cell growth, telomerase activity and DNA repair in human breast cancer MCF-7 cells.

p16INK4a, a cell cycle inhibitor that inhibits cyclin-dependent kinase 4 (cdk4) and cdk6, has been found as the tumor suppressor gene and is frequently deleted, methylated or mutated in many malignancies. Since p16INK4a is also a key element controlling cellular senescence and other functions, we hypothesized that p16INK4a induced tumor suppression may not be limited to the inhibition of cdks. To investigate the role of p16INK4a in tumor suppression and the potential interaction between p16INK4a and other cellular controlling elements, such as telomerase activity and DNA repair ability, the full-length of p16INK4a cDNA was cloned into a retroviral vector and introduced into human breast cancer MCF-7 cells that were previously demonstrated to harbor homozygous deletions of the p16INK4a gene. Stable expression of p16INK4a suppressed the malignant phenotype in MCF-7 cells, including cell proliferation, anchorage-independent growth, G1/G0 cell cycle arrest, and the blockage of pRB phosphorylation. In addition, expression of p16INK4a suppressed telomerase activity and restored the telomere shortening process, and decreased cell DNA repair ability and sensitized cells to the DNA damage reagent. Our data suggest that the wild-type p16INK4a plays an important role in suppression of tumor malignancy, not only by inhibiting cell proliferation through cell cycle arrest, but also by inhibiting other cellular controlling mechanisms, such as telomerase activity and DNA repair capacity.

Adenocarcinoma↗

PubMed2002

TCRbeta transmembrane tyrosines are required for pre-TCR function.

The pre-TCR promotes thymocyte development in the alphabeta lineage. Productive rearrangement of the TCRbeta locus triggers the assembly of the pre-TCR, which includes the pTalpha chain and CD3 epsilongammadeltazeta subunits. This complex receptor signals the up-regulation of CD4 and CD8 expression, thymocyte proliferation/survival, and the cessation of TCRbeta rearrangements (allelic exclusion). In this study, we investigate the function of two conserved tyrosine residues located in the TCRbeta chain transmembrane region of the pre-TCR. We show that replacement of both tyrosines with alanine and expression of the mutant receptor in RAG-1(null) thymocytes prevents surface expression and abolishes pre-TCR function relative to wild-type receptor. Replacement of both tyrosines with phenylalanines (YF double mutant) generates a complex phenotype in which thymocyte survival and proliferation are severely disrupted, differentiation is moderately disrupted, and allelic exclusion is unaffected. We further show that the YF double mutant receptor is expressed on the cell surface and associates with pTalpha and CD3epsilon at the same level as does wild-type TCRbeta, while association of the YF double mutant with CD3zeta is slightly reduced relative to wild type. These data demonstrate that pre-TCR signaling pathways leading to proliferation and survival, differentiation, and allelic exclusion are differently sensitive to subtle mutation-induced alterations in pre-TCR structure.

Amino Acid Sequence↗

PubMed2002

A mammalian expression system for rapid production and purification of active MAP kinase phosphatases.

Expression of enzymatically active mammalian proteins in Escherichia coli can proven to be a challenging task due to poor solubility, improper folding, and lack of adequate posttranslational modification. Expression of mammalian proteins using baculovirus or yeast systems is time-consuming and may also be subject to inadequate modification. In order to overcome these technical difficulties, we have developed a mammalian expression system for the convenient subcloning of cDNA fragments, high-level expression, and one-step purification of enzymatically active proteins. The mammalian expression vector pEBG that expresses glutathione S-transferase fusion proteins was modified to create an SrfI restriction site in the multiple cloning site. The protein coding sequences of MAP kinase phosphatase-1 (MKP-1), MAP kinase phosphatase-2 (MKP-2), and the tumor suppressor PTEN were PCR-amplified using Pfu DNA polymerase and cloned into the SrfI site through SrfI digestion-coupled ligation. The resulting plasmids were transiently transfected into 293T cells using FuGENE 6 transfection reagent. Forty eight hours after transfection, cells were harvested and bioactive recombinant proteins were purified by glutathione-Sepharose beads. Protein yield, which ranged from 200 to 700 microg, was more than adequate for biochemical studies. The usefulness of this versatile system for studying protein function and its potential application for proteomics research are discussed.

Animals↗

PubMedDate not supplied

Metabolic labeling of human primary retinal pigment epithelial cells for accurate comparative proteomics.

Metabolic labeling was evaluated, using both 13C6-Arg and 13C6, 15N2-Lys amino acids, for a primary human retinal pigment epithelial cell (hRPE) culture prepared from an autopsy eye of an 81 year old donor. Satisfactory incorporation (>90%) was achieved with both stable isotope labeled amino acids after four passages (roughly 7 population doublings). The degree of incorporation was found to be efficient with both amino acids as well as in different proteins. The presence of 10% whole serum in the culture medium did not interfere with the incorporation of the exogenous stable isotope labeled amino acids. Metabolic labeling of these human primary retinal pigment epithelial cells was further tested to quantify protein ratios between proliferating and resting cells using a combination of 2-DG and MALDI-TOF-TOF/MS analysis. Using computational data processing and analysis, we obtained accurate protein ratio measurement for every single identified protein (156 proteins) in the 2-Dg array. Of these 156 proteins, 12 proteins were found significantly increased in dividing versus resting cells by at least a factor of 1.5 while 13 other proteins were found increased in resting versus dividing cells by at least the same fold. Most of these differentially expressed proteins are directly involved in cell proliferation, protein synthesis, and actin-remodeling and differentiation.

Aged↗