William C. Hyun

INHIBITION OF PROTEASOME ACTIVITY BLOCKS CELL CYCLE PROGRESSION AT SPECIFIC PHASE BOUNDARIES IN AFRICAN TRYPANOSOMES

Proteasomes are one of the cellular complexes controlling protein degradation from archaebacteria to mammalian cells. We recently purified and characterized the catalytic core of the proteasome, the 20S form, from Trypanosoma brucei, a flagellated protozoa which causes African trypanosomiasis. To identify the role of proteasomes in African trypanosomes, we used lactacystin, a specific inhibitor of proteasome activity. Lactacystin showed potent inhibition of the activity of 20S proteasomes purified from both bloodstream and procyclic (insect) forms of T. brucei (IC50 = 1 microM). It also inhibited proliferation of T. brucei cells in culture assays, with 1 microM inhibiting growth of bloodstream forms, whereas 5 microM was required to block proliferation of procyclic forms. Analysis of the DNA content of these cells by flow cytometry showed that 5 microM lactacystin arrested procyclic cells in the G2 + M phases of the cell cycle. Fluorescence microscopy revealed that most of the cells had one nucleus and one kinetoplast each, indicating that the cells had replicated their DNA, but failed to undergo mitosis. This suggests that transition from G2 to M phase was blocked. On the other hand, incubation of bloodstream forms with 1 microM lactacystin led to arrest of 30-35% of the cell population in G1 and 55-60% of the cells in G2, indicating that both transition from G1 to S and from G2 to M were blocked. These observations were also confirmed by using another inhibitor of proteasome, N-carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal (LLnV), which arrested procyclic forms in G2, and bloodstream forms in both G1 and G2. These results suggest that proteasome activity is essential for driving cell cycle progression in T. brucei, and that proteasomes may control cellular functions differently in bloodstream and procyclic forms of T. brucei.

EGF‐induced redistribution of erbB2 on breast tumor cells: Flow and image cytometric energy transfer measurements

erbB2, a member of the epidermal growth factor (EGF) receptor-type tyrosine kinase receptor family, is overexpressed in breast carcinomas with poor prognosis. We examined the cell surface association of this receptor with itself and with other cell surface proteins by the Förster-type fluorescence resonance energy transfer using whole antibodies and Fab fragments. We found that erbB2 molecules homoassociate in unstimulated SK-BR-3, BT474 and BT474-M (a metastatic version of the parent BT474 line) breast tumor cells, and that the interaction was enhanced by EGF treatment in suspensions of SK-BR-3 and BT474-M cells. BT474 cells (with low EGF receptor expression) and attached SK-BR-3 cells do not respond to EGF. Image microscopic energy transfer measurements found considerable pixel-by-pixel heterogeneity in the homoassociation state of erbB2. In accordance with the EGF-induced redistribution of erbB2, EGF receptor was found to be in close proximity to erbB2 in FRET measurements. By labeling different epitopes on erbB2 and the lipid bilayer, we were able to prepare an epitope map of erbB2 molecule. Our data suggest the existence of dynamic cell surface patterns of erbB2 and point to functions fulfilled by these molecular complexes.

Lymphoid follicles are generated in high-grade cervical dysplasia and have differing characteristics depending on HIV status.

The exact role of the mucosal immune response in the pathogenesis of human papillomavirus (HPV)-related premalignant and malignant diseases of the genital tract is poorly understood. We used immunohistochemical analysis to characterize immune cells in normal cervix (N = 21), HIV-negative high-grade dysplasia (N = 21), and HIV-positive high-grade dysplasia (N = 30). Classical germinal centers were present in 4.7% of normal cervix, 33% of high-grade lesions from HIV-negative women, and 3.3% of high-grade lesions from HIV-positive women (P = 0.003). HPV16 E7 antigen was detected in a subset of germinal centers, indicating that the secondary immune response was directed in part against HPV. Lymphoid follicles were present in 9.5% of normal cervix, 57% of HIV-negative high-grade dysplasia, and 50% of HIV-positive high-grade dysplasia (P = 0.001 normal versus high-grade). A novel type of lymphoid aggregate, consisting predominantly of CD8(+) T cells, was detected in 4.8% of normal cervix, 0% of HIV-negative high-grade dysplasia, and 40% of HIV-positive high-grade dysplasia (P < 0.001). The recurrence rate of high-grade dysplasia within one year was significantly higher in women with such CD8(+) T cell-dominant aggregates (P = 0.02). In summary, the types of lymphoid follicle in lesions from HIV-positive women were significantly different from those from HIV-negative women, and these differences are associated with the worse clinical outcome in HIV-positive women.

Analyses of brain tumor cell lines confirm a simple model of relationships among fluorescence in situ hybridization, DNA index, and comparative genomic hybridization

Several techniques are commonly used for genetic analysis of interphase nuclei. Flow cytometry assays the distribution of DNA content in populations of nuclei stained with a DNA‐specific fluorochrome. Fluorescence in situ hybridization (FISH) quantifies the number of copies of a specific DNA sequence in single nuclei. Comparative genomic hybridization (CGH) assesses the relative copy number of DNA sequences throughout a test genome by comparing the signal intensities of test and reference DNA samples hybridized to a template of normal metaphase chromosomes. In principle, there are specific relationships among data obtained from these measurements, and combined measurements should provide a more comprehensive view of the sample that is analyzed. We applied these three techniques to nine brain tumor cell lines and find that a model of CGH that includes unsuppressed repeat sequences describes the data well. We estimate that up to 35% of the fluorescence intensity in well‐blocked CGH preparations may not represent unique sequences. Taking these factors into account, our results are, in general, mutually consistent, and highlight issues critical for interpreting CGH preparations. Genes Chromosomes Cancer 20:311‐319, 1997.

Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins

The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry.

Distinctive Features of Angiogenesis and Lymphangiogenesis Determine Their Functionality during De novo Tumor Development

Blood and lymphatic vasculature are essential components of all organs, responsible for maintaining organ fluid dynamics and tissue homeostasis. Although both vessel systems are composed of similar lineages of endothelial cells whose crude functions include fluid and cell transport, each system also possesses distinctive physiologic properties, enabling their distinctive functions in tissues. The role of hematogenous vasculature and development of angiogenic blood vessels during cancer development is well established; however, the role of lymphangiogenesis and structural/functional alterations occurring within lymphatic vessels during cancer development are incompletely understood. To assess premalignant versus malignant alterations in blood and lymphatic vasculature associated with squamous epithelial skin carcinogenesis, we assessed architectural and functional features of both vascular systems using a mouse model of de novo carcinoma development. We report that, as vasculature acquires angiogenic and/or lymphangiogenic properties, angiogenic blood vessels become leaky in premalignant tissue and at peripheries of carcinomas, where enlarged lymphatic capillaries efficiently drain increased tissue fluid, thereby maintaining tissue hemodynamics. In contrast, central regions of carcinomas exhibit elevated tissue fluid levels, compressed lymphatic lumina, and decreased vascular leakage, thus indicating impaired hemodynamics within solid tumors. Together, these data support the notion that therapeutic delivery of anticancer agents is best realized in premalignant tissues and/or at the peripheries of solid tumors where hemodynamic forces support drug delivery. Strategies to normalize intratumoral hemodynamics would therefore enhance therapeutic delivery to otherwise poorly accessible central regions of solid tumors.

Spectral imaging in preconception/preimplantation genetic diagnosis of aneuploidy: multicolor, multichromosome screening of single cells.

Purpose:Our purpose was to evaluate the utility of spectral imaging for multicolor, multichromosome enumeration in human interphase cell nuclei.Methods:Chromosome-specific probes labeled with different fluorochromes or nonfluorescent haptens were obtained commercially or prepared in-house. Metaphase spreads, interphase lymphocytes, or blastomeres cells were hybridized with either 7 or 11 distinctly different probes. Following 46 hr of hybridization, slides were washed and detected using either a filter-based quantitative image processing system (QUIPS) developed in-house or a commercial spectral imaging system.Results:The filter-based fluorescence microscope system is preferred for simultaneous detection of up to seven chromosome targets because of its high sensitivity and speed. However, this approach may not be applicable to interphase cells when 11 or more targets need to be discriminated. Interferometer-based spectral imaging with a spectral resolution of approximately 10 nm allows labeling of chromosome-specific DNA probes with fluorochromes having greatly overlapping emission spectra. This leads to increases in the number of fluorochromes or fluorochrome combinations available to score unambiguously chromosomes in interphase nuclei.Conclusions:Spectral imaging provides a significant improvement over conventional filter-based microscope systems for enumeration of multiple chromosomes in interphase nuclei, although further technical development is necessary in its application to embryonic blastomeres. When applied to preconception/preimplantation genetic diagnosis, presently available probes for spectral imaging are expected to detect abnormalities responsible for 70–80% of spontaneous abortions caused by chromosomal trisomies.

Quantification of neurotoxicity and identification of cellular subsets in a three‐dimensional brain model

Imaging of cells in a large intact three-dimensional tissue remains difficult. Quantification and identification of cell damage in a mixed culture system has been limited by the inability of fluorescent probes to discriminate types of cellular death and penetrate tissue more that 100 microm thick. We have investigated several probes in combination with neural cell-specific antibodies to quantify cell damage in the presence of several toxins. Acridine orange and ethidium bromide were excellent for determination of cell viability, death by necrosis, or apoptosis in thick brain tissue aggregates. Calcein and ethidium homodimer were effective on live/ dead stains, and the Syto dyes 11 and 13 worked well for quantification of all cells in the brain aggregate model. By using these combinations of dyes in conjunction with confocal microscopy, we were able to quantify neural cell damage without disrupting the three-dimensional environment.

Simultaneous flow cytometric analyses of enhanced green and yellow fluorescent proteins and cell surface antigens in doubly transduced immature hematopoietic cell populations

BACKGROUND Cell transduction with multiple genes offers opportunities to investigate specific gene interactions on cell function. Detection of multiple transduced genes in hematopoietic cells requires strategies to combine measurements of gene expression with phenotypic cell discriminants. We describe simultaneous flow cytometric detection of two green fluorescent protein (GFP) variants in immunophenotypically defined human hematopoietic subpopulations using only a minor physical adjustment to a standard FACSCalibur. METHODS The accuracy and sensitivity of enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP) detection in mixtures of transduced and nontransduced PG13 packaging cells were evaluated by flow cytometry. Retroviral vectors encoding EGFP or EYFP were used to transduce CD34(+) hematopoietic cells derived from umbilical cord blood. The transduction efficiency into subpopulations of hematopoietic cells was measured using multivariate flow cytometry. RESULTS A bicistronic retroviral vector containing the EGFP and puromycin N-acetyltransferase (pac) genes afforded brighter EGFP signals in transduced cells than a retroviral vector encoding a pac-EGFP fusion protein. The sensitivity of detecting EGFP and EYFP-expressing cells among a background of nonexpressing cells was 0.01% and 0.05%, respectively. EGFP or EYFP was expressed in up to 95% of CD34(+) DR(-) or CD34(+) 38(-) subpopulations in cord blood 48 h posttransduction. Simultaneous transduction with EGFP and EYFP viral supernatants (1:1 mixture) led to coexpression of both GFP variants in 15% of CD34(+) DR(-) and 20% of CD34(+) 38(-) cells. CONCLUSIONS These results demonstrate simultaneous detection of EGFP and EYFP in immunophenotypically discriminated human hematopoietic cells. This technique will be useful to quantify transduction of multiple retroviral constructs in discriminated subpopulations.

Modulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by chemotherapy in thyroid cancer cell lines.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many human cancer cells but not in normal cells. Thyroid cancer cells, however, appear to be relatively resistant to TRAIL-induced apoptosis. We therefore investigated the effect of chemotherapy on TRAIL-induced apoptosis in thyroid cancer cells. We used six thyroid cancer cell lines: TPC-1, FTC-133, FTC-236, FTC-238, XTC-1, and ARO82-1. We used flow cytometry to measure apoptosis, dimethyl-thiazol-diphenyltetrazolium bromide (MTT) assay to measure antiproliferation effects and Western blot to determine the expression of Bcl family proteins. Troglitazone, paclitaxel, geldanamycin, and cycloheximide were used for pretreatment. We used the Students t test and analysis of variance (ANOVA) for statistical analysis. All thyroid cancer cell lines, except the TPC-1 cell line, were resistant to TRAIL, and growth inhibition was less than 20% at concentration of 800 ng/mL of TRAIL. In both TPC-1 (TRAIL-sensitive) and FTC-133 (TRAIL-resistant) thyroid cancer cell lines, pretreatment with troglitazone, cycloheximide, and paclitaxel enhanced TRAIL-induced cell death significantly but pretreatment with geldanamycin did not. There were no significant changes in Bcl-2, Bcl-xl, and Bax protein expression after troglitazone treatment. In conclusion, TRAIL in combination with troglitazone, paclitaxel, and cycloheximide induces apoptosis in thyroid cancer cells at suboptimal concentrations that cannot be achieved using TRAIL alone.