Jun-Ping Liu

Polymorphism of human cytochrome P450 enzymes and its clinical impact

Pharmacogenetics is the study of how interindividual variations in the DNA sequence of specific genes affect drug response. This article highlights current pharmacogenetic knowledge on important human drug-metabolizing cytochrome P450s (CYPs) to understand the large interindividual variability in drug clearance and responses in clinical practice. The human CYP superfamily contains 57 functional genes and 58 pseudogenes, with members of the 1, 2, and 3 families playing an important role in the metabolism of therapeutic drugs, other xenobiotics, and some endogenous compounds. Polymorphisms in the CYP family may have had the most impact on the fate of therapeutic drugs. CYP2D6, 2C19, and 2C9 polymorphisms account for the most frequent variations in phase I metabolism of drugs, since almost 80% of drugs in use today are metabolized by these enzymes. Approximately 5–14% of Caucasians, 0–5% Africans, and 0–1% of Asians lack CYP2D6 activity, and these individuals are known as poor metabolizers. CYP2C9 is another clinically significant enzyme that demonstrates multiple genetic variants with a potentially functional impact on the efficacy and adverse effects of drugs that are mainly eliminated by this enzyme. Studies into the CYP2C9 polymorphism have highlighted the importance of the CYP2C9*2 and *3 alleles. Extensive polymorphism also occurs in other CYP genes, such as CYP1A1, 2A6, 2A13, 2C8, 3A4, and 3A5. Since several of these CYPs (e.g., CYP1A1 and 1A2) play a role in the bioactivation of many procarcinogens, polymorphisms of these enzymes may contribute to the variable susceptibility to carcinogenesis. The distribution of the common variant alleles of CYP genes varies among different ethnic populations. Pharmacogenetics has the potential to achieve optimal quality use of medicines, and to improve the efficacy and safety of both prospective and currently available drugs. Further studies are warranted to explore the gene-dose, gene-concentration, and gene-response relationships for these important drug-metabolizing CYPs.

TELOMERASE IS CONTROLLED BY PROTEIN KINASE CALPHA IN HUMAN BREAST CANCER CELLS

Telomerase, a specialized RNA-directed DNA polymerase that extends telomeres of eukaryotic chromosomes, is repressed in human somatic tissues and becomes activated during tumor progression in most human cancers. To date, little is known about how telomerase is activated and controlled in cancer, although activation is thought to be involved in cancer cell immortalization. Here, we report that human telomerase-associated protein 1 (hTEP1) and the telomerase catalytic subunit (human telomerase reverse transcriptase (hTERT)) are phosphoproteins and that their phosphorylation is a prerequisite for the activation of telomerase in intact human breast cancer cells. Identified by hTEP1 peptide affinity chromatography, protein kinase Cα mediates the phosphorylation of hTEP1 and hTERT and induces a marked increase in telomerase activity. Thus, phosphorylation of hTEP1 and hTERT by protein kinase Cα represents an essential step in the generation of a functional telomerase complex in the initiation and maintenance of telomerase activity in human cancer.

Protein Phosphatase 2A Inhibits Nuclear Telomerase Activity in Human Breast Cancer Cells

Most cancer cells have increased levels of telomerase activity implicated in cell immortalization. Activation of telomerase, a ribonucleoprotein complex, catalyzes the elongation of the ends of mammalian chromosomal DNA (telomeres), the length of which regulates cell proliferation. Currently, how telomerase is regulated in cancer is not yet established. The present study shows that telomerase activity is regulated by protein phosphorylation in human breast cancer cells. Incubation of cell nuclear telomerase extracts with protein phosphatase 2A (PP2A) abolished the telomerase activity; in contrast cytoplasmic telomerase activity was unaffected, and protein phosphatases 1 and 2B were ineffective. Inhibition of telomerase activity by PP2A was both concentration- and time-dependent and was prevented by the protein phosphatase inhibitor okadaic acid. In addition, nuclear telomerase inhibited by PP2A was reactivated by endogenous protein kinase(s) in the presence of ATP, but not in the presence of ATPγS. Furthermore, telomerase activity in cultured human breast cancer PMC42 cells was stimulated by okadaic acid, consistent with a role for PP2A in the regulation of telomerase activity in intact cells. These findings suggest that protein phosphorylation reversibly regulates the function of telomerase and that PP2A is a telomerase inhibitory factor in the nucleus of human breast cancer cells.

TERT regulates cell survival independent of telomerase enzymatic activity.

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, plays a pivotal role in the maintenance of telomeres and cell proliferation. Here we report that down-regulation of hTERT induces apoptosis independently of telomerase enzymatic activity in human breast cancer cells. Expression of a hTERT mutant lacking telomerase activity rescues the cells with lowered telomerase without inducing cell death. With similar patterns of subcellular distribution to that of the tumor suppressor protein p53 during mitosis, hTERT interacts with p53 and poly(ADP-ribose) polymerase (PARP). Decreasing p53 expression in intact cells worsens, and increasing p53 prevents, cell death induced by lowering hTERT. Thus, hTERT maintains cell survival and proliferation via both telomerase enzymatic activity-dependent telomere lengthening and enzymatic activity-independent intermolecular interactions involving p53 and PARP.

Structure, function, regulation and polymorphism and the clinical significance of human cytochrome P450 1A2

Human CYP1A2 is one of the major CYPs in human liver and metabolizes a number of clinical drugs (e.g., clozapine, tacrine, tizanidine, and theophylline; n > 110), a number of procarcinogens (e.g., benzo[a]pyrene and aromatic amines), and several important endogenous compounds (e.g., steroids). CYP1A2 is subject to reversible and/or irreversible inhibition by a number of drugs, natural substances, and other compounds. The CYP1A gene cluster has been mapped on to chromosome 15q24.1, with close link between CYP1A1 and 1A2 sharing a common 5′-flanking region. The human CYP1A2 gene spans almost 7.8 kb comprising seven exons and six introns and codes a 515-residue protein with a molecular mass of 58,294 Da. The recently resolved CYP1A2 structure has a relatively compact, planar active site cavity that is highly adapted for the size and shape of its substrates. The architecture of the active site of 1A2 is characterized by multiple residues on helices F and I that constitutes two parallel substrate binding platforms on either side of the cavity. A large interindividual variability in the expression and activity of CYP1A2 has been observed, which is largely caused by genetic, epigenetic and environmental factors (e.g., smoking). CYP1A2 is primarily regulated by the aromatic hydrocarbon receptor (AhR) and CYP1A2 is induced through AhR-mediated transactivation following ligand binding and nuclear translocation. Induction or inhibition of CYP1A2 may provide partial explanation for some clinical drug interactions. To date, more than 15 variant alleles and a series of subvariants of the CYP1A2 gene have been identified and some of them have been associated with altered drug clearance and response and disease susceptibility. Further studies are warranted to explore the clinical and toxicological significance of altered CYP1A2 expression and activity caused by genetic, epigenetic, and environmental factors.

Cholesterol involvement in the pathogenesis of neurodegenerative diseases

Cholesterol, an essential component of cell membranes, plays an important role in the maintenance of cellular homeostasis and transmembrane communication within and between cellular compartments. In the brain that contains the highest levels of cholesterol in the body, cholesterol traffic occurs between nerve cells and between intracellular organelles in neurons to subserve normal brain function. Whereas glial cells produce the largest quantities of cholesterol, neurons also acquire cholesterol synthesized by astrocytes. The intracellular organelle endosomes and lysosomes receive and distribute cholesterol through the endocytic and retrograde transport pathways. However, deregulated cholesterol trafficking appears to be involved in the pathogenesis of Alzheimers disease (AD), Parkinsons disease (PD) and Niemann-Pick disease type C (NPC) diseases. Under the pathological conditions of these neurodegenerative diseases, aberrant molecular interactions or particular depositions of cholesterol have been observed as critical causes to precipitate neuronal cell death. Here, we review the recent advances in terms of the role of cholesterol in healthy brain and molecular mechanisms of cholesterol involvement in AD, PD and NPC diseases. We discuss the different lines of evidence supporting different models of anomalous intracellular cholesterol trafficking with emphasis on cholesterol interactions with alpha-synuclein, NPC1 and NPC2 in AD, PD and NPC.

Transforming Growth Factor β Suppresses Human Telomerase Reverse Transcriptase (hTERT) by Smad3 Interactions with c-Myc and the hTERT Gene

Telomerase underpins stem cell renewal and proliferation and is required for most neoplasia. Recent studies suggest that hormones and growth factors play physiological roles in regulating telomerase activity. In this report we show a rapid repression of the telomerase reverse transcriptase (TERT) gene by transforming growth factor β (TGF-β) in normal and neoplastic cells by a mechanism depending on the intracellular signaling protein Smad3. In human breast cancer cells TGF-β induces rapid entry of Smad3 into the nucleus where it binds to the TERT gene promoter and represses TERT gene transcription. Silencing Smad3 gene expression or genetically deleting the Smad3 gene disrupts TGF-β repression of TERT gene expression. Expression of the Smad3 antagonist, Smad7, also interrupts TGF-β-mediated Smad3-induced repression of the TERT gene. Mutational analysis identified the Smad3 site on the TERT gene promoter, mediating TERT repression. In response to TGF-β, Smad3 binds to c-Myc; knocking down c-Myc, Smad3 does not bind to the TERT gene, suggesting that c-Myc recruits Smad3 to the TERT promoter. Thus, TGF-β negatively regulates telomerase activity via Smad3 interactions with c-Myc and the TERT gene promoter. Modifying the interaction between Smad3 and TERT gene may, thus, lead to novel strategies to regulate telomerase.

Ets2 Maintains hTERT Gene Expression and Breast Cancer Cell Proliferation by Interacting with c-Myc

Human telomerase reverse transcriptase (hTERT) underlies cancer cell immortalization, and the expression of hTERT is regulated strictly at the gene transcription. Here, we report that transcription factor Ets2 is required for hTERT gene expression and breast cancer cell proliferation. Silencing Ets2 induces a decrease of hTERT gene expression and increase in human breast cancer cell death. Reconstitution with recombinant hTERT rescues the apoptosis induced by Ets2 depression. In vitro and in vivo analyses show that Ets2 binds to the EtsA and EtsB DNA motifs on the hTERT gene promoter. Mutation of either Ets2 binding site reduces the hTERT promoter transcriptional activity. Moreover, Ets2 forms a complex with c-Myc as demonstrated by co-immunoprecipitation and glutathione S-transferase pulldown assays. Immunological depletion of Ets2, or mutation of the EtsA DNA motif, disables c-Myc binding to the E-box, whereas removal of c-Myc or mutation of the E-box also compromises Ets2 binding to EtsA. Thus, hTERT gene expression is maintained by a mechanism involving Ets2 interactions with the c-Myc transcription factor and the hTERT gene promoter, a protein-DNA complex critical for hTERT gene expression and breast cancer cell proliferation.

GAPDH: a common enzyme with uncommon functions.

1. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) has long been recognized as an important enzyme for energy metabolism and the production of ATP and pyruvate through anaerobic glycolysis in the cytoplasm.

Establishment, immortalisation and characterisation of pteropid bat cell lines

Background Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. Methodology/Findings Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. Conclusions/Significance The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.