Cells alkaline elution

DIA, DNA single strand breaks, cross-links or related damage, LYS mouse lymphoma cells, alkaline elution in vitro... 

Iqbal. DNA chain growth during replication of asynchronous L1210 cells. Alkaline elution of large DNA segments from cells lysed on filters. Biochemistry 13 4134-4139, 1974. 

Boekelheide, K. (1993) Sertoli cell toxicants. In Russell, L.D. Griswold, M.D., eds. The Sertoli Cell, Clearwater, FL, Cache River Press, pp. 551-575 Bove, J.L., Dalven, P. Kukreja, V.P (1978) Airborne di-butyl and di(2-ethylhexyl)phthalate at three New York City air sampling stations. Int. J. environ. Chem., 5, 189-194 Bradley, M.O. (1985) Measurement of DNA single-strand breaks by alkaline elution in rat hepatocytes. Prog. Mutat. Res., 5, 353-357... 

Phenol did not induce DNA single-strand breaks in mouse lymphoma L5178Y cells. It was reported in abstracts that phenol induced DNA strand breaks in mouse lymphoma cells, as measured by the alkaline unwinding technique followed by elution through hydroxyapatite (Garberg Bolesfoldi, 1985), but that it did not induce strand breaks, as measured by the alkaline elution technique, in rat germ-cell DNA after either single or multiple dose treatments (Skare Schrotel, 1984). 

As already indicated, strand breaks can be detected and measured by alkaline sucrose-gradient centrifugation and by alkaline elution. Both procedures have been applied to cells from intact animals, as well as to in vitro systems. 

Alkaline elution of large DNA fragments from cells lysed on filters was devised by Kohn and colleagues215 216 and has been applied to the study of damaging effects on DNA of carcinogens given to animals in vivo. As with the... 

Kohn, K.W., L.C. Erickson, R.A. Ewig, and C.A. Friedman. Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry 15 4629-4637,... 

Alkaline elution techniques In one type of method the cellular DNA is denatured in alkali followed by hydroxyapatite chromatography which separates single stranded DNA from double stranded DNA (Britten and Kohne, 1965). The production of single-stranded DNA is related to the number of SSBs in the DNA which act as unwinding points. As an example the effects of UV-irradiation on the integrity of mammalian cell DNA has been studied using this approach (Collins, 1977). Initially mammalian cells in culture are extensively labelled with [3H]thymidine (see Adams, this series, 1980). 

As was the case for the alkaline elution technique described above, the DNA of the cultured cell has to be first extensively labelled with [3H]thymidine (see Adams, this series, 1980). 

DNA ISC formation can be measured relatively easily in plasmid DNA using an agarose gel based assay (10) and ISC formation and its repair can be measured in intact cells using the technique of alkaline elution (//). Although sensitive enough to measure ISC at pharmacologically relevant doses, alkaline... 

The technique is reproducible, more sensitive than methods such as alkaline elution, requires fewer cells and has the important advantage that analysis can be made at the single cell level. It is therefore possible to determine heterogeneity of ISC formation and its repair in a cell population. The method is applicable to studies with cultured cell lines, human lymphocytes and solid tumor tissue. Correlations can therefore be made between both the level of ISCs and the efficiency of their repair with both inherent and acquired resistance to DNA crosslinking agents in vitro and in vivo. 

Kohn, K. W., and Grimek-Ewig, R. A. (1973). Alkaline elution analysis, a new approach to the study of DNA single-strand intemiptions in cells. Cancer Res 33, 1849-1853. 

FIGURE 3. Alkaline elution studies in SK-OV-3 cells after exposure to 100 pM [Au(OAc>2(damp)] or 100 pM cisplatin. DNA cross-link formation is indicated by reduced flow through the filter, as is the case with DNA from the cisplatin-treated cells (a). DNA strand breaks are indicated by enhanced flow through the filter, as demonstrated by DNA from irradiated cells (b). untreated control, O 100 pM cisplatin, 100 pM [Au(OAc)2(damp)], Irradiated control... 

Figure 5, Repair of ci DDP-iruiuced DNA-protein crosslinks and DNA inter-strand crosslinks in human fibroblasts during holding in the nondividing state. Stationary cultures were treated with 40 fjjnol cvs-DDP, and after various periods of holding in the nondividing state, crosslinking was measured by alkaline elution (U DNA-protein crosslinks and O, DNA interstrand crosslinks) or by estimation of renaturable DNA in cell lysates (%, DNA interstrand crosslinks).

The Assessment of DNA Crosslinking by Alkaline Elution. DNA damage, that is, interstrand crosslinks, DNA-protein crosslinks, and strand breaks, was determined using the alkaline elution technique (7, ). Cells labeled with C-thymidine for 20-24 hr were deposited on a membrane filter and lysed with a detergent-containing solution. An alkaline solution (pH 12.1-12.2) was then slowly pumped through the filter, and fractions were collected to determine the rate of release of DNA from the filter. For assay of crosslinks, the cells were exposed to x-ray at 0 C prior to deposition on the filter. In order to improve quantitation, control cells labeled with H-thymidine and x-irradiated at 0 were mixed with the experimental Relabeled cells prior to deposition on the filters. The elution of H-DNA serves as an internal reference for normalization of the elution of C-DNA. 

Figure 3, The alkaline elution kinetics of DNA from L1210 cells treated with cis-DDP (10 jM.) for 1 h followed by 12 h incubation in a drug-free medium, L1210 cells were labeled with C-thymidine for 20 h and were either cis-DDP treated (A A) or untreated (%, O). (Reproduced with permission from Ref. 20, Copyright 1980, Academic Press.)...

How can such an approach be extended in the future Erickson et al have shown with their work in human cells one important step The results of studies using human material as a target for cis-DDP may reveal differences from results obtained in rodent systems These results may have more bearing on clinical problems (, ) Erickson e al have also developed a fluorometric alkaline elution assay requiring no labeled DNA... 

Fig. 1. Alkaline elution profiles of WIL-2 cells after 1 mM MMS (30 min) and 4 h growth with increasing concentrations of SAB...

Fig. 3. DNA single-strand breaks in HeLa cells following treatment with 50 ijM DMS benzamide. HeLa cells were labeled overnight with [ C]-thymidine, the medium was changed and the cells were incubated with 50 juAf DMS. Benzamide was added to the culture 1 h after DMS. Aliquots for the determination of DNA fragmentation by the alkaline elution technique [15] were removed at the times indicated...

Logarithmically growing Friend cells were labeled with [ C]-thymidine (0.03 juCi ml ) for 16 h, then diluted into fresh medium containing test chemicals. Subsequently these cells were removed at various times following the treatment and analyzed for DNA strand breaks using the alkaline elution method [18]. 

Strand breaks. DMSO did not induce DNA strand breaks after the first 24 h of induction although a 24 h exposure resulted in a twentyfold increase in benzidine-positive cells (Fig. 1). Analysis of DNA strand breaks by alkaline elution, showed no difference between controls and DMSO-treated cells at 0, 2, 4, 6, 8 and 12 h (data not shown). Addition of 10 mM SAB together with DMSO (Fig. 1) or the addition of SAB separately did not cause an increase in DNA strand breaks (data not shown). Sodium butyrate and N -methylnicotinamide did not induce single-strand breaks in DNA at 12 or 24 h of incubation (data not shown). Inducers of FEL differentiation and inhibitors of poly(ADP-ribose) polymerase did not cause detectable DNA strand breaks under the experimental conditions used in these studies. 

SAB and analyzed by alkaline elution. Control cells had no treatment (O), and 100 rad of gamma rays from Cesium ( ). The elution profiles of Friend cell DNA treated DMSO for 0, 2, 4, 6, 8 and 12 h were not different from the controls... 

Fig. 2. Dimethyl sulfate causes DNA single-strand breaks in Friend cells that are potentiated by SAB. [ Cl-thymidine labeled Friend cells, treated with 50 yM dimethyl sulfate for 30 min, were centrifuged and resuspended in fresh media with or without SAB. Samples were removed at 0, S and 7 h after treatment and analyzed by alkaline elution. Numbers refer to h and + to presence of SAB...

Enhanced rate of DNA strand break repair by hyper-expression of poly(ADP-ribose) polymerase cDNA. In the data in Fig. 1 above, we established that a significantly increased potential for poly(ADP-ribosyl)ation would exist around 2 days after transfection with pcD-12. We decided to use this time window to directly test whether poly(ADP-ribosyl)ation is involved in DNA repair by measuring the resealing of X-ray induced DNA strand breaks (8). Cos cells were prelabeled with [i C]thymidine for two days, after which cells were either mock transfected, transfected with pcC-12 or carried through without any transfection protocol. After 44 hr, the cells were irradiated with 2000 rads on ice and immediately allowed to repair at 37 C. Cells were maintained on ice until assayed by alkaline elution. Unrepaired SSB (single strand breaks) m experimental DNA were compared to the control gamma-ray induced SSB and expressed as the radiation dose that would produce Rad-equivalent breaks. The alkaline elution method used was the short assay to detect SSB up to 3000 SSB rad-equivalents. 

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