Vitamin E and EDTA Improve the Efficacy of Hypothermosol-Implication of Apoptosis.

The emergence of engineered tissues and new cell strains has called for the need to explore solutions that can be used to store these cells and tissues in a state of near suspended animation without using cryopreservation. The ability of the hypothermic solutions ViaSpan (DuPont Merck Pharmaceutical Company, Wilmington, DE), HypoThermosol (Cryomedical Sciences, Rockville, MD), HypoThermosol supplemented with either ethylenediaminetetraacetic acid (EDTA) or Vitamin E and HypoThermosol supplemented with apoptosis protease inhibitors were tested for their abilities to cold-protect Madin Darby Canine Kidney (MDCK) cells at 4 degrees C. Alamar Blue, a nontoxic metabolic indicator was used to measure cell viability. The order of cold protection was HypoThermosol with Vitamin E and EDTA > HypoThermosol with Vitamin E > HypoThermosol with EDTA > HypoThermosol > ViaSpan > Dulbecco's Modified Eagle's Medium (DMEM). Membrane integrity tests supported the Alamar Blue data that EDTA and Vitamin E conferred a benefit to the cold-storage capabilities of HypoThermosol. MDCK cells that died subsequent to 1 to 6 days cold-storage detached from the substratum and their DNA was harvested after being placed at 37 degrees C. This DNA was compared to DNA retrieved from adherent cells in the same cultures that survived the cold-storage regime. Gel electrophoresis of cells dying due to 1 to 4 days of cold-storage showed a DNA ladder indicating that cells died through apoptosis, programmed cell death. Dead cells harvested at 5 to 6 days of cold storage, however, had randomly cleaved DNA indicative of necrotic cell death. HypoThermosol supplemented with apoptosis protease inhibitors was better able to cold-protect cells than the base HypoThermosol. These data suggest that the inhibition of apoptosis should be considered in the future cold-storage formulations.

Cryosurgical Modeling: Sequence of Freezing and Cytotoxic Agent Application Affects Cell Death.

Cryosurgery is now being used to eradicate a variety of tumors, including those that are found in the kidney. Because it is well known that cancer treatments are often most effective when combined with adjunctive therapies, we developed a model kidney cell culture system to determine: (1) if 5-fluorouracil (5-FU), a compound known to be a poor kidney tumor chemotherapeutic agent, can be more effective when combined with cryosurgery; and (2) how kidney cells die through freezing-induced injury that might occur during cryosurgery. The DNA isolated from kidney cells that died during an exposure to -15 degrees C showed nonrandom cleavage when separated on an agarose gel, indicating that cell death was attributable, in part, to apoptosis, whereas DNA isolated from kidney cells that died during exposure to -75 degrees C showed random cleavage, indicating that cell death was attributable, in part, to necrosis. The apoptotic protease inhibitor, caspase 1 inhibitor V, was able to prevent freezing-induced cell death, supporting the idea that apoptosis may be a mechanism of cell death in the periphery of the iceball created by cryosurgical procedures in vivo. Because 5-FU is known to induce apoptosis, this drug was used in combination with various freezing regimes to determine if the combination might be a better method of killing cells than either treatment alone. Whereas the addition of 5-FU at the same time or 2 days after freezing resulted in a synergistic lethal effect, many cells survived this combination treatment. When cells were treated with 5-FU 2 days prior to freezing, however, there was an apparent complete loss of viability. The reason may be that, by an unknown mechanism, freezing enhances the ability of 5-FU-treated cells to move more effectively through the apoptosis pathway. Kidney tumors may have robust antiapoptotic mechanisms that make them refractory to 5-FU, but these mechanisms might be overridden by freezing. In either case, the data suggest that chemotherapy may be more effective when followed by cryosurgery.

Restriction-modification systems in the marine, aerobic, nitrogen-fixing unicellular cyanobacterium Cyanothece sp. ATCC 51142.

Cyanothece sp. ATCC 51142 possesses six host restriction-modification systems (HRMS), and these HRMS were designated as Csp68KI to Csp68KVI. So far, four restriction enzymes have been characterized biochemically, and the other two were deduced based on the cloning of corresponding DNA methyltransferase genes M. Csp68KIV and M. Csp68KV from the Cyanothece genome. Csp68KI, Csp68KII, Csp68KIII, Csp68KIV, Csp68KV, and Csp68KVI are isoschizomers of AvaII, AsuII, AvaIII, MspI, HaeIII, and FnuDII, respectively. The cleavage specificities for Csp68KI, Csp68KII, Csp68KIII, and Csp68KVI were characterized. The Cyanothece restriction enzymes showed different temperature and salt requirements for their optimal activity. For example, the restriction enzyme Csp68KII functions optimally at 50 degreesC and Csp68KIII requires higher salt for its activity.