No abstract

To better understand the basis for the difference in radiosensitivity between the variant murine leukemic lymphoblast cell lines L5178Y-R (resistant) and L5178Y-S (sensitive), the production and repair of DNA damage after X irradiation were measured by the DNA alkaline and neutral elution techniques. The initial yield of single-strand DNA breaks and the rates of their repair were found to be the same in both cell lines by the DNA alkaline elution technique. Using the technique of neutral DNA elution, L5178Y-S cells exhibited slightly increased double-strand breakage immediately after irradiation, most significantly at lower doses (i.e., less than 10 Gy). Nevertheless, even at doses that yielded equal initial double-strand breakage of both cell lines, the survival of L5178Y-S cells was significantly less than that of L5178Y-R cells. When the technique of neutral DNA elution was employed to measure the kinetics of DNA double-strand break repair, both cell lines exhibited biphasic fast and slow components of repair. However, the double-strand repair rate was much lower in the radiosensitive L5178Y-S cells than in the L5178Y-R cells (T1/2 of 60 vs 16 min). This difference was more pronounced in the fast-repair component. These results suggest that the repair of double-strand DNA breaks is an important factor determining the radiosensitivity of L5178Y cells.

Pulsed-field gel electrophoresis (PFGE) is one of the most sensitive methods for detecting DNA double-strand breaks in mammalian cells. However, it has been observed that constant-field gel electrophoresis (CFGE), when optimized, can detect breaks with equal efficiency. The migration of DNA from the well and the separation of DNA molecules according to size appear to be different processes; only the latter requires the application of PFGE. CFGE is very sensitive and can detect DNA damage produced by less than 5 Gy of radiation. Low voltage (ca. 0.6 V/cm) during electrophoresis appears to be essential for the migration of the largest fraction of DNA from the agarose plug containing the cells; the electrophoresis run time, cell density in the plug, agarose concentration, nature of detergent and extent of radiolabelling are less important. It is concluded that CFGE is equally sensitive but more rapid and economical than PFGE for the measurement of DNA damage.

alpha(4)beta(1)-Integrin (very late antigen-4 (VLA-4)) mediates cell adhesion to cell surface ligands (VCAM-1). Binding of VLA-4 to VCAM-1 initiates rolling and firm adhesion of leukocytes to vascular endothelium followed by the extravasation into the tissue. VLA-4-dependent adhesion plays a key role in controlling leukocyte adhesive events. Small molecules that bind to the integrin ligand-binding site and block its interaction with natural ligands represent promising candidates for treatment of several diseases. Following a flow cytometric screen for small molecule discovery, we took advantage of a conformationally sensitive anti-beta(1)-integrin antibody (HUTS-21) and a small LDV-containing ligand (LDV-FITC) with known affinity to study binding affinities of several known and recently discovered integrin ligands. We found that binding of the LDV-containing small molecule induced exposure of HUTS-21 epitope and that the EC(50) for antibody binding was equal to previously reported K(d) for fluorescent LDV (LDV-FITC). Thus, binding of HUTS-21 can be used to report ligand-binding site occupancy. We studied binding of two known integrin ligands (YLDV and TR14035), as well as of two novel compounds. EC(50) values for HUTS-21 binding showed good correlation with K(i)s determined in the competition assay with LDV-FITC for all ligands. A docking model suggests a common mode of binding for the small molecule VLA-4 ligands. This novel approach described here can be used to determine ligand-binding affinities for unlabeled integrin ligands, and can be adapted to a high-throughput screening format for identification of unknown integrin ligands.

We investigated the role of initial DNA and chromosome damage in determining the radiosensitivity difference between the variant murine leukemic lymphoblast cell lines L5178Y-S (sensitive) and L5178Y-R (resistant) and the difference in cell cycle-dependent variations in radiosensitivity of L5178Y-S cells. We measured initial DNA damage (by the neutral filter elution method) and chromosome damage (by the premature chromosome condensation method) and compared them with survival (measured by cloning) for both cell lines synchronized in G1 or G2 phase of the cell cycle (by centrifugal elutriation) and irradiated with low doses of X rays (up to 10 Gy). The initial yield of DNA and chromosome damage in G2 L5178Y-S cells was almost twice that in G1 L5178Y-S cells and G1 or G2 L5178Y-R cells. In all cases DNA damage expressed as relative elution corresponded with chromosome damage (breaks in G1 chromosomes, breaks and gaps in G2 chromosomes). Also we found that the initial DNA and chromosome damage did not determine cell age-dependent radiosensitivity variations in L5178Y-S cells, as there was less initial damage in the more sensitive G1 phase than in the G2 phase. L5178Y-R cells showed only small changes in survival or initial yield of DNA and chromosome damage throughout the cell cycle. Because survival and initial damage in sensitive and resistant cells irradiated in G2 phase correlated, the difference in radiosensitivity between L5178Y-S and L5178Y-R cells might be determined by initial damage in G2 phase only.