Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls

BACKGROUND: Telomeres containing noncoding DNA repeats at the end of the chromosomes are essential for chromosomal stability and are implicated in regulating the replication and senescence of cells. The gradual loss of telomere repeats in cells has been linked to aging and tumor development and methods to measure telomere length are of increasing interest. At least three methods for measuring the length of telomere repeats have been described: Southern blot analysis and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q-FISH) or flow cytometry (flow-FISH). Both Southern blot analysis and Q-FISH have specific limitations and are time-consuming, whereas the flow-FISH technique requires relatively few cells (10(5)) and can be completed in a single day. A further advantage of the flow-FISH method is that data on the telomere length from individual cells and subsets of cells (lymphocytes and granulocytes) can be acquired from the same sample. In order to obtain accurate and reproducible results using the flow-FISH technique, we systematically explored the influence of various steps in the protocol on telomere length values and established an acceptable range for the most critical parameters. METHODS: Isolated leukocytes from whole blood are denatured by heat and 70%/75% formamide, then hybridized with or without a telomere-specific fluorescein isothiocyante (FITC)-conjugated peptide nucleic acid probe (PNA). Unbound telomere PNA is washed away, the DNA is counterstained, and telomere fluorescence is measured on a flow cytometer using an argon ion laser (488 nm) to excite FITC. For each sample, duplicates of telomere PNA-stained and unstained tubes are analyzed. RESULTS: Cell counts and flow-FISH telomere length measurements were performed on leukocytes and thymocytes of humans and other species. Leukocyte suspensions were prepared by two red blood cell lysis steps with ammonium chloride. Optimal denaturation of DNA was achieved by heating at 85-87 degrees C for 15 min in a solution containing 70%/75% formamide. Hybridization was performed at room temperature with a 0.3 microg/ml telomere-PNA probe for at least 60-90 min. Unbound telomere-PNA probe was diluted at least 4,000-40,000 times with wash steps containing 70%/75% formamide at room temperature. LDS 751 and DAPI were suitable as DNA counterstains as they did not show significant interference with telomere length measurement. CONCLUSIONS: The use of flow-FISH for telomere length measurements in nucleated blood cells requires tight adherence to an optimized protocol. The method described here can be used to determine rapidly the telomere length in subsets of nucleated blood cells.     

Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner

In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3(+) related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.     

Role of SODD in regulation of tumor necrosis factor responses

Signaling from tumor necrosis factor receptor type 1 (TNFR1) can elicit potent inflammatory and cytotoxic responses that need to be properly regulated. It was suggested that the silencer of death domains (SODD) protein constitutively associates intracellularly with TNFR1 and inhibits the recruitment of cytoplasmic signaling proteins to TNFR1 to prevent spontaneous aggregation of the cytoplasmic death domains of TNFR1 molecules that are juxtaposed in the absence of ligand stimulation. In this study, we demonstrate that mice lacking SODD produce larger amounts of cytokines in response to in vivo TNF challenge. SODD-deficient macrophages and embryonic fibroblasts also show altered responses to TNF. TNF-induced activation of NF-kappaB is accelerated in SODD-deficient cells, but TNF-induced c-Jun N-terminal kinase activity is slightly repressed. Interestingly, the apoptotic arm of TNF signaling is not hyperresponsive in the SODD-deficient cells. Together, these results suggest that SODD is critical for the regulation of TNF signaling.     

CD4+ Th1 and CD8+ type 1 cytotoxic T cells both play a crucial role in the full development of contact hypersensitivity

The role of CD4(+) vs CD8(+) T cells in contact hypersensitivity (CHS) remains controversial. In this study, we used gene knockout (KO) mice deficient in CD4(+) or CD8(+) T cells to directly address this issue. Mice lacking either CD4(+) or CD8(+) T cells demonstrated depressed CHS responses to dinitrofluorobenzene and oxazolone compared with wild-type C57BL/6 mice. The depression of CHS was more significant in CD8 KO mice than in CD4 KO mice. Furthermore, in vivo depletion of either CD8(+) T cells from CD4 KO mice or CD4(+) T cells from CD8 KO mice virtually abolished CHS responses. Lymph node cells (LNCs) from hapten-sensitized CD4 and CD8 KO mice showed a decreased capacity for transferring CHS. In vitro depletion of either CD4(+) T cells from CD8 KO LNCs or CD8(+) T cells from CD4 KO LNCs resulted in a complete loss of CHS transfer. LNCs from CD4 and CD8 KO mice produced significant amounts of IFN-gamma, indicating that both CD4(+) and CD8(+) T cells are able to secrete IFN-gamma. LNCs from CD8, but not CD4, KO mice were able to produce IL-4 and IL-10, suggesting that IL-4 and IL-10 are mainly derived from CD4(+) T cells. Intracellular cytokine staining of LNCs confirmed that IFN-gamma-positive cells consisted of CD4(+) (Th1) and CD8(+) (type 1 cytotoxic T) T cells, whereas IL-10-positive cells were exclusively CD4(+) (Th2) T cells. Collectively, these results suggest that both CD4(+) Th1 and CD8(+) type 1 cytotoxic T cells are crucial effector cells in CHS responses to dinitrofluorobenzene and oxazolone in C57BL/6 mice.     

Transgenic overexpression of beta(2)-adrenergic receptors in airway epithelial cells decreases bronchoconstriction

Airway epithelial cells express beta(2)-adrenergic receptors (beta(2)-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of beta(2)-ARs to airway epithelium of transgenic (CCSP-beta(2)-AR) mice, thereby mimicking agonist activation of receptors only in these cells. In situ hybridization confirmed that transgene expression was confined to airway epithelium, and autoradiography showed that beta(2)-AR density in CCSP-beta(2)-AR mice was approximately twofold that of nontransgenic (NTG) mice. Airway responsiveness measured by whole body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline (ED(200)) was greater for CCSP-beta(2)-AR than for NTG mice (345 +/- 34 vs. 157 +/- 14 mg/ml; P < 0.01). CCSP-beta(2)-AR mice were also less responsive to ozone (0.75 ppm for 4 h) because enhanced pause in NTG mice acutely increased to 77% over baseline (P < 0.05) but remained unchanged in the CCSP-beta(2)-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED(200) for ozone-exposed CCSP-beta(2)-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell beta(2)-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of beta-agonists results from activation of receptors on both epithelial and smooth muscle cells.     

The protein kinase C cascade regulates recruitment of matrix metalloprotease 9 to podosomes and its release and activation

Podosomes are transient cell surface structures essential for degradation of extracellular matrix during cell invasion. Protein kinase C (PKC) is involved in the regulation of podosome formation; however, the roles of individual PKC isoforms in podosome formation and proteolytic function are largely unknown. Recently, we reported that PDBu, a PKC activator, induced podosome formation in normal human bronchial epithelial cells. Here, we demonstrate that phorbol-12,13-dibutyrate (PDBu)-induced podosome formation is mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes. Interestingly, although blocking atypical PKCζ did not affect PDBu-induced podosome formation, it significantly reduced matrix degradation at podosomes. Inhibition of PKCζ reduced recruitment of matrix metalloprotease 9 (MMP-9) to podosomes and its release and activation. Downregulation of MMP-9 by small interfering RNA (siRNA) or neutralization antibody also significantly reduced matrix degradation. The regulatory effects of PKCζ on matrix degradation and recruitment of MMP-9 to podosomes were PKCζ kinase activity dependent. PDBu-induced recruitment of PKCζ and MMP-9 to podosomes was blocked by inhibition of novel PKC with rottlerin or PKCδ siRNA. Our data suggest that multiple PKC isozymes form a signaling cascade that controls podosome formation and dynamics and MMP-9 recruitment, release, and activation in a coordinated fashion.