Christopher J. Donahue

Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer

Fas ligand (FasL) is produced by activated T cells and natural killer cells and it induces apoptosis (programmed cell death) in target cells through the death receptor Fas/Apo1/CD95 (ref. 1). One important role of FasL and Fas is to mediate immune-cytotoxic killing of cells that are potentially harmful to the organism, such as virus-infected or tumour cells. Here we report the discovery of a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds to FasL and inhibits FasL-induced apoptosis. The DcR3 gene was amplified in about half of 35 primary lung and colon tumours studied, and DcR3 messenger RNA was expressed in malignant tissue. Thus, certain tumours may escape FasL-dependent immune-cytotoxic attack by expressing a decoy receptor that blocks FasL.

Activation of apoptosis by Apo-2 ligand is independent of FADD but blocked by CrmA

A new member of the tumor necrosis factor (TNF) cytokine family, designated Apo-2 ligand (Apo-21) [1] or TRAIL [2], has been shown recently to induce apoptosis in various tumor cell lines; however, its biological role is unknown. Here, we show that Apo-21, activated apoptosis in T-cell-enriched cultures of peripheral blood lymphocytes stimulated by interleukin-2 (IL-2), but not in unstimulated cells. This finding suggests that, like Fas/Apo-1 ligand and TNF [3-5], Apo-2L may play a role in regulating post-stimulation apoptosis of mature lymphocytes. Studies on the mechanism of Apo-2L action demonstrated marked membrane blebbing, a hallmark of apoptosis, within a few minutes of the addition of Apo-2L to tumor cells. Ectopic expression of a dominant negative mutant of FADD, a cytoplasmic protein that mediates death signalling by Fas/Apo-1 and by TNF receptor type 1 (TNFR1) [6-9], inhibited the induction of apoptosis by anti-Fas/Apo-1 antibody, but had little effect on Apo-2L function. In contrast, expression of CrmA, a cowpox virus-derived inhibitor of the Ced-2-like proteases ICE [10] and CPP32/Yama [11,12], blocked the induction of apoptosis by either Apo-2L or anti-Fas/Apo-1 antibody. These results suggest that Apo-2L activates a rapid, FADD-independent pathway to trigger a cell-death programme that requires the function of cysteine proteases such as ICE or CPP32/Yama.

Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultues

Temperature reduction in CHO cell batch culture may be beneficial in the production of recombinant protein and in maintenance of viability. The effects on cell cycle, apoptosis and nucleotide pools were studied in cultures initiated at 37°C and temperature shifted to 30 °C after 48 hours. In control cultures maintained at 37 °C, viable cells continued to proliferate until the termination of the culture, however, temperature reduction caused a rapid decrease in the percent of cells in S phase and accumulation of cells in G-1. This was accompanied by a concurrent reduction in U ratio (UTO/UDP-GNAc), previously shown to be a sensitive indicator of growth rate. Culture viability was extended following temperature shift, as a result of delayed onset of apoptosis, however, once initiated, the rate and manner of cell death was similar to that observed at 37 °C. All nucleotide pools were similarly degraded at the time of apoptotic cell death. Temperature reduction to 30 °C did not decrease the energy charge of the cells, however, the overall rate of metabolism was reduced. The latter may be sufficient to extend culture viability via a reduction in toxic metabolites and/or limitation of nutrient deprivation. However, the possibility remains that the benefits of temperature reduction in terms of both viability and productivity are more directly associated with cultures spending extended time in G-1.

Apoptosis in CHO cell batch cultures: examination by flow cytometry.

Chinese hamster ovary cells grown under conditions which are optimal for the production of a genetically engineered protein in batch culture, lose significant viability shortly after entering the stationary phase. This cell death was investigated morphologically and was found to be almost exclusively via apoptosi. Furthermore, cells were analyzed by flow cytometry using a fluorescent DNA end-labeling assay to label apoptotic cells, in conjunction with cell cycle analysis using propidium iodide. Apoptotic cells could be detected by this method, and by the radioactive end-labeling of extracted DNA, on all days of culture from day 1 to day 7; however, the degree of apoptotic cell death increased dramatically when the cells entered the stationary phase, rising to 50–60% of the total cell number at the termination of the culture. Flow cytometric analysis showed that the majority of cells underwent apoptosis whilst in G1/G0 and formed an apoptotic population with high DNA FITC end-labeling and hypodiploid propidium iodide binding. Additionally, the ability or inability to secrete specific protein products did not appear to interfere with the development of the apoptotic population with time.

Aequorea green fluorescent protein: simultaneous analysis of wild-type and blue-fluorescing mutant by flow cytometry.

Aequorea green fluorescent protein (GFP) has been expressed in a variety of cell lines and host organisms. A recent report (Heim et al.: Proc Natl Acad Sci USA 91:12501-12504, 1994) has documented that a GFP mutant with a single amino acid substitution (tyrosine 66 to histidine; Y66H-GFP) elicits altered spectral properties. Whereas wild-type GFP emits with a maximum at approximately 509 nm (green fluorescence), Y66H-GFP fluoresces with a maximum at approximately 448 nm (blue fluorescence). In this study we employed available argon and krypton ion laser lines to investigate the impact of laser excitation wavelength on the detection of Y66H-GEP by flow cytometry. Using transiently transfected 293 cells, a cellular subpopulation with elevated blue fluorescence was detectable with excitation at 407 nm, but not with ultraviolet (UV), 458 nm, or 488 nm excitation. The blue-fluorescing cells were further documented to express Y66H-GFP by immunoblot analysis of sorted cells. Finally, we demonstrated the simultaneous analysis of both wild-type and Y66H-GFP in cotransfected 293 cells using 407 nm excitation while collecting blue fluorescence at 460 +/- 20 nm (Y66H-GFP) and green fluorescence at 525 +/- 25 nm (wild-type GFP). These studies illustrate the potential for assessing differential gene expression by simultaneously analyzing multiple GFP species with multiparameter flow cytometry.

Multicolor immunofluorescence and flow cytometry utilizing cascade blue to purify murine hematopoietic stem cells from fetal liver and bone marrow

BACKGROUND Here we demonstrate the utility of cascade blue (CB), to purify hematopoietic stem cells by flow cytometry. Multicolor immunofluorescence and the sensitivity (signal-to-noise) of the fluorochromes are essential for the identification and isolation of rare stem cell populations. METHODS We isolated hematopoietic stem cells utilizing a 407 nm laser line to excite CB and propidium iodide (PI) in combination with FITC, PE, and Red670 which were excited at 488 nm. RESULTS CB is maximally excited using a 407 nm laser line, when compared to UV or 413 nm excitation. The increase in sensitivity of CB at 407 nm can be contributed to higher absorption of CB and a reduction of autofluorescence at this excitation wavelength (Ropp et al.: Cytometry 21: 309-317, 1995). CONCLUSIONS Despite the fact that the CB antibody conjugate has a tendency to adhere specifically to a B cell subpopulation in bone marrow, we nevertheless could purify stem cells by using CB for the detection and elimination of lineage positive cells. Isolated stem cells from mouse fetal liver (Lin-CD34(+)Sca-1(+)c-Kit(high)) and adult bone marrow (Lin-CD34(-/low)Sca-1(+)c-Kit(+)) were transplanted into lethally irradiated mice, and the sorted stem cells had the ability to efficiently repopulate all mature hematopoietic lineages in recipient mice.