Richard Y. Zhao

Blinded, Multicenter Comparison of Methods To Detect a Drug-Resistant Mutant of Human Immunodeficiency Virus Type 1 at Low Frequency

ABSTRACT We determined the abilities of 10 technologies to detect and quantify a common drug-resistant mutant of human immunodeficiency virus type 1 (lysine to asparagine at codon 103 of the reverse transcriptase) using a blinded test panel containing mutant-wild-type mixtures ranging from 0.01% to 100% mutant. Two technologies, allele-specific reverse transcriptase PCR and a Ty1HRT yeast system, could quantify the mutant down to 0.1 to 0.4%. These technologies should help define the impact of low-frequency drug-resistant mutants on response to antiretroviral therapy.

Up-regulation of 14-3-3ζ in Lung Cancer and Its Implication as Prognostic and Therapeutic Target

A functional genomic approach integrating microarray and proteomic analyses done in our laboratory has identified 14-3-3zeta as a putative oncogene whose activation was common and driven by its genomic amplification in lung adenocarcinomas. 14-3-3zeta is believed to function in cell signaling, cycle control, and apoptotic death. Following our initial finding, here, we analyzed its expression in lung tumor tissues obtained from 205 patients with various histologic and stage non-small cell lung cancers (NSCLC) using immunohistochemistry and then explored the effects of specific suppression of the gene in vitro and in a xenograft model using small interfering RNA. The increased 14-3-3zeta expression was positively correlated with a more advanced pathologic stage and grade of NSCLCs (P = 0.001 and P = 0.006, respectively) and was associated with overall and cancer-specific survival rates of the patients (P = 0.022 and P = 0.018, respectively). Down-regulation of 14-3-3zeta in lung cancer cells led to a dose-dependent increased sensitivity to cisplatin-induced cell death, which was associated with the inhibition of cell proliferation and increased G2-M arrest and apoptosis. The result was further confirmed in the animal model, which showed that the A549 lung cancer cells with reduced 14-3-3zeta grew significantly slower than the wild-type A549 cells after cisplatin treatment (P = 0.008). Our results suggest that 14-3-3zeta is a potential target for developing a prognostic biomarker and therapeutics that can enhance the antitumor activity of cisplatin for NSCLC.

Genetic Deletions in Sputum as Diagnostic Markers for Early Detection of Stage I Non–Small Cell Lung Cancer

Purpose: Analysis of molecular genetic markers in biological fluids has been proposed as a powerful tool for cancer diagnosis. We have characterized in detail the genetic signatures in primary non–small cell lung cancer, which provided potential diagnostic biomarkers for lung cancer. The aim of this study was to determine whether the genetic changes can be used as markers in sputum specimen for the early detection of lung cancer. Experimental Design: Genetic aberrations in the genes HYAL2, FHIT, and SFTPC were evaluated in paired tumors and sputum samples from 38 patients with stage I non–small cell lung cancer and in sputum samples from 36 cancer-free smokers and 28 healthy nonsmokers by using fluorescence in situ hybridization. Results:HYAL2 and FHIT were deleted in 84% and 79% tumors and in 45% and 40% paired sputum, respectively. SFTPC was deleted exclusively in tumor tissues (71%). There was concordance of HYAL2 or FHIT deletions in matched sputum and tumor tissues from lung cancer patients (r = 0.82, P = 0.04; r = 0.84, P = 0.03), suggesting that the genetic changes in sputum might indicate the presence of the same genetic aberrations in lung tumors. Furthermore, abnormal cells were found in 76% sputum by detecting combined HYAL2 and FHIT deletions whereas in 47% sputum by cytology, of the cancer cases, implying that detecting the combination of HYAL2 and FHIT deletions had higher sensitivity than that of sputum cytology for lung cancer diagnosis. In addition, HYAL2 and FHIT deletions in sputum were associated with smoking history of cancer patients and smokers (both P < 0.05). Conclusions: Tobacco-related HYAL2 and FHIT deletions in sputum may constitute diagnostic markers for early-stage lung cancer.

Viral infections and cell cycle G2/M regulation

ABSTRACTProgression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast (Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyr15) on Cdc2, which is phosphorylated by Wee1 kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two well-characterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins, which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-1) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest. Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.

Roles of HIV-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions

ABSTRACTActive host-pathogen interactions take place during infection of human immunodeficiency virus type 1 (HIV-1). Outcomes of these interactions determine the efficiency of viral infection and subsequent disease progression. HIV-infected cells respond to viral invasion with various defensive strategies such as innate, cellular and humoral immune antiviral mechanisms. On the other hand, the virus has also developed various offensive tactics to suppress these host cellular responses. Among many of the viral offensive strategies, HIV-1 viral auxiliary proteins (Tat, Rev, Nef, Vif, Vpr and Vpu) play important roles in the host-pathogen interaction and thus have significant impacts on the outcome of HIV infection. One of the best examples is the interaction of Vif with a host cytidine deaminase APOBEC3G. Although specific roles of other auxiliary proteins are not as well described as Vif-APOBEC3G interaction, it is the goal of this brief review to summarize some of the preliminary findings with the hope to stimulate further discussion and investigation in this exhilarating area of research.

Single-Cell Fluorescence Imaging Using Metal Plasmon-Coupled Probe 2: Single-Molecule Counting on Lifetime Image

Multiple Alexa Fluor 647-conjugated concanavalin A (conA) molecules were covalently bound to a single 20 nm silver particle to synthesize metal plasmon-coupled probes (PCPs). The fluorescence images were recorded by scanning confocal microscopy in both intensity and lifetime. The brightness of PCPs was 30-fold brighter than those of free conA and the lifetime of PCPs was shortened dramatically. PCPs were used to label T-lymphocytic ( PM1) cells. The emission spots by PCPs bound on the cell surfaces were separated clearly from the cell images by autofluorescence due to the brighter signal and shorter lifetime of PCPs. The emission spots by PCPs were also scanned in three dimensions to count the distribution of bound fluorophores on the cell surfaces. The metal-associated fluorophores thus are suggested using as novel molecular imaging agents to quantify the components and describe their distributions on the cell surfaces.

Enhanced Fluorescence Images for Labeled Cells on Silver Island Films

Silver island films (SIFs) were deposited on glass substrates to serve as supports. T-Lymphocytic (PM1) cell lines were labeled by Alexa Fluor 680-dextran conjugates on the membranes or by YOYO in the nuclei. The fluorescence images of the cell lines were recorded in the emission intensity and lifetime using scanning confocal microscopy. The fluorescence signals by the fluorophores bound on the cell membranes were enhanced significantly by SIF supports as compared with those on the glass. In addition to the increase in the intensity, there was a dramatic shortening of the emission lifetime. In contrast to the Alexa Fluor 680 fluorophores on the membranes, the YOYO fluorophores intercalated in the cell nuclei were not influenced significantly by the silver islands. This result can be interpreted by an effect of the distance on coupling between the fluorophores and metal particles: the fluorophores on the cell membranes are localized within, but the fluorophores in the cell nuclei are beyond the region of metal-enhanced fluorescence. Thus, the metal supports can be used to improve the detection sensitivity for target molecules on cell surfaces when they are fluorescently labeled.

Vpr-Host Interactions During HIV-1 Viral Life Cycle

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is a multifunctional viral protein that plays important role at multiple stages of the HIV-1 viral life cycle. Although the molecular mechanisms underlying these activities are subject of ongoing investigations, overall, these activities have been linked to promotion of viral replication and impairment of anti-HIV immunity. Importantly, functional defects of Vpr have been correlated with slow disease progression of HIV-infected patients. Vpr is required for efficient viral replication in non-dividing cells such as macrophages, and it promotes, to some extent, viral replication in proliferating CD4+ T cells. The specific activities of Vpr include modulation of fidelity of viral reverse transcription, nuclear import of the HIV-1 pre-integration complex, transactivation of the HIV-1 LTR promoter, induction of cell cycle G2 arrest and cell death via apoptosis. In this review, we focus on description of the cellular proteins that specifically interact with Vpr and discuss their significance with regard to the known Vpr activities at each step of the viral life cycle in proliferating and non-proliferating cells.

Fluorescent Avidin-Bound Silver Particle: A Strategy for Single Target Molecule Detection on a Cell Membrane

Cy5-avidin conjugate-bound silver nanoparticles were prepared as a fluorescence molecular reagent for the cell imaging. Compared with the metal-free avidin conjugate, the avidin-metal complex was observed to display a stronger emission intensity, shorter lifetime, and better photostability. The avidin-metal complexes were conjugated with the biotin-sites on the surfaces of PM1 cell lines, and the cell images were recorded using scanning confocal microscopy. It was noticed that the avidin-metal complexes bound on the cell surfaces could be identified as the isolated emission spots distinct from the cellular autofluorescence. The emission intensity over the cell image was increased with an increase of the number of avidin-metal complexes on the cell surface but the lifetime was decreased. A quantitative regression curve was achieved between the amount of avidin-metal complex on the cell surface and the emission intensity or lifetime over the entire cell image. On the basis of this curve, we expect to develop an approach that can be used to quantify the amount of target molecules on the cell surfaces using the cell intensity and lifetime images at the single cell level.

Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

BackgroundCell cycle G2 arrest induced by HIV-1 Vpr is thought to benefit viral proliferation by providing an optimized cellular environment for viral replication and by skipping host immune responses. Even though Vpr-induced G2 arrest has been studied extensively, how Vpr triggers G2 arrest remains elusive.ResultsTo examine this initiation event, we measured the Vpr effect over a single cell cycle. We found that even though Vpr stops the cell cycle at the G2/M phase, but the initiation event actually occurs in the S phase of the cell cycle. Specifically, Vpr triggers activation of Chk1 through Ser345 phosphorylation in an S phase-dependent manner. The S phase-dependent requirement of Chk1-Ser345 phosphorylation by Vpr was confirmed by siRNA gene silencing and site-directed mutagenesis. Moreover, downregulation of DNA replication licensing factors Cdt1 by siRNA significantly reduced Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest. Even though hydroxyurea (HU) and ultraviolet light (UV) also induce Chk1-Ser345 phosphorylation in S phase under the same conditions, neither HU nor UV-treated cells were able to pass through S phase, whereas vpr-expressing cells completed S phase and stopped at the G2/M boundary. Furthermore, unlike HU/UV, Vpr promotes Chk1- and proteasome-mediated protein degradations of Cdc25B/C for G2 induction; in contrast, Vpr had little or no effect on Cdc25A protein degradation normally mediated by HU/UV.ConclusionsThese data suggest that Vpr induces cell cycle G2 arrest through a unique molecular mechanism that regulates host cell cycle regulation in an S-phase dependent fashion.