Nucleoids sedimentation

Fig. 7. Effect of ethidium bromide on the sedimentation of nucleoids from cells incubated benzamide. HeLa cells in suspension culture were incubated with 5 mM benzamide. 4 h after ben-zamide addition cells were sedimented, suspended in saline, and applied to isokinetic sucrose gradients (containing bis-benzimide and increasing amounts of ethidium bromide) overlaid with a lysis buffer containing Triton X-100. 15 min later the gradients were centrifuged for 30 min at 16,000 rpm. The sedimentation distance was determined by yisual inspection under UV light. Inset Effect of benzamide on the sedimentation of nucleoids from benzamide-treated cells. HeLa cells were incubated for 4 h with increasing amounts of benzamide and th the nucleoid sedimentation rate was determined in bis-benzimide containing sucrose gradients as described above...

Fig. la,b. Nucleoid sedimentation analysis. HeLa cells were grown for 24 h at 37°C in the presence of [6- H] thymidine at 1 fiCi mf (sp.act. 26 Ci mmol" ). The medium was then removed and replaced with fresh medium (a) or with medium containing 100 mA/ methotrexate (b) and incubation continued for 1 h. Subsequent nucleoid sedimentation analysis (6000 rpm for 30 min at 20°C, SW50.1 rotor) was as described by Cook and Brazell [3]. Fractions were collected from the bottom of gradients and precipitated onto GF/C filters for radioactivity determination. Sedimentation is from right to left... 

Within 2 h of addition of concanavalin A (Con A) to mouse splenic lymphocytes, there was repair of 3200 strand breaks per diploid genome (Table 1) DNA strand breaks were assayed by means of fluorescence analysis of DNA unwinding [7]. This technique was used to demonstrate the same phenomenon in pig peripheral blood lymphocytes within 5 h (Table 1). This technique does not differentiate between alkali labile bonds and frank breaks however, this argument does not apply to the work of Johnstone and Williams [3] who used the method of nucleoid sedimentation in neutral gradients. Thus in human lymphocytes, at least, frank breaks exist in resting cells. 

Fig. 2a,b. Estimation of DNA strand breaks by the nucleoid sedimentation procedure, a Sedimentation of nucleoids from HL-60 cells either exposed to 7-radiation b treated with the indicated concentration of DMS for 15 min... 

Fig. 4. Repair of DNA strand breaks induced by DMS. Undifferentiated ( , O) or differentiated (A, A) cells were treated with either 10 juA/ closed symbols) or 50 juAf open symbols) DMS for 15 min (defined as time 0 of repair). At indicated times cells were removed and their nucleoid sedimentation rate was determined...

Fig. 6a,b. Transient formation of DNA strand breaks during DMSO-induced differentiation of HL-60 cells. Nucleoid sedimentation rate in uninduced control cultures ( ) and in DMSO-induced cultures (A, , ). a Control (O) or DMSO-treated cultures ( ) in the presence of 2 mM 3-methoxybenz-amide. b Control (Q), and DMSO-treated cultures (0) in the presence of 2 mM 3-methoxybenzoic acid... 

In spite of the notion that tumor promotion does not involve mutation of the genome, this process must nevertheless be able to markedly influence nuclear processes like cell proliferation and differentiation. In certain systems, promotors like 12-0-tetradecanoyl-phorbol-13-acetate (TPA) or benzoylperoxide (BPO) can induce DNA fragmentation [23]. This may, however, not be a general phenomenon. When 3T3 cells are incubated with TPA (up to 50 mg mP ) for 3 h no significant change in nucleoid sedimentation... 

Fig. 1. Benzoylperoxide-induced activation of poly(ADPR) polymerase in 3T3 cells. Cells were incubated for 3 h with increasing concentrations of benzoylperoxide and analyzed for DNA fragmentation (nucleoid sedimentation). NAD content, and poly(ADPR) polymerase activity in permeabilized cells DNase...

Changes in DNA supercoiling were analyzed using nucleoid sedimentation as previously described and are shown in Fig. 1 (2). Human granulocyte-macrophage preciusor cells were prepared from bone marrow samples (obtained with consent from healthy volunteers) by density fractionation and adherence (2). Cells were exposed to two inducers of granulocytic differentiation (retinoic acid and G-CSF) and two inducers of monocytic differentiation (phorbol myristate acetate and vitamin D3). Treatment with differentiation inducers and culture conditions are described elsewhere (2). 

Fig. 1. Migration ratios on nucleoid sedimentation gradients following treatment with differentiation inducers, in the presence (dashed line) and absence of 5 mM 3-methoxybenzamide A) 10- M PMA B) 10-6 M RET C) 10- M Vit D3 D) G-CSA (S0% umbilical cord conditioned medium). Results are means of five, six, five and two independent experiments for A-D respectively. With the monocytic inducers (PMA and Vit D3) there was an increase in sedimentation. In contrast, with the granulocytic inducers (RET, G-CSA) there was a transient retardation of sedimentation. The increased sedimentation rate induced by the monocytic inducers was abolished by 3-MB.

DNA strand breaks were monitored by nucleoid sedimentation (16). DMA induced DNA strand break levels were increased dramatically in the presence of SAB (Fig. 4). Although there was a dose dependent increase in 6TG induced DNA strand breaks, no further increase was observed in the presence of SAB. However, caution must be exercised in the interpretation of these results, since at least some 6TG induced DNA strand breaks have been shown to be protein crosslinked (18), and therefore alkaline elution studies are required to confirm these results. Note that although we can detect breaks using concentrations of DMS that yield 100% survival, doses of 6TG that reduce survival to less than 10% must be used. 

Fig. 10a,b. The sedimentation of nucleoids from differentiating chick muscle cells, a The lysis mixture contained 1.0 M NaCl ( ) or 1.96 M NaCl ( ), or 1.95 M NaCl plus 5 Mg/ml ethidium bromide ( ). b The effect of ethidium bromide. The lysis mixture contained 1.95 M NaCl and the indicated concentration of ethidium bromide. 24 h cultures 72 h cultures... 

This inference is also drawn from several experiments with ethidium bromide. In the presence of 5 jUg of ethidium bromide the sedimentation of nucleoids from 24 h cultures is reduced from 1.0 to 0.67 (Fig. 10a). However, the same concentration of ethidium bromide has a much smaller effect on the sedimentation at 96 h, which is only reduced from 0.56 to 0.46. In nucleoids from 24 h cultures, increasing concentrations of ethidium bromide induce the biphasic effect characteristic of DNA molecules under topological constraints (Fig. 10b), whereas nucleoids from 72 h cultures are... 

Fig. la,b. Sedimentation of nucleoids from cells treated with 10 jtiAf DMS in the presence or absence of 3AB. Cells were incubated with or without 5 mM 3AB for 30 min before addition of 10 fiM DMS. Samples were removed at various times after DMS addition and nucleoids prepared and run (Cook and Brazell). Results are expressed as the ratio of migration of DMS treated to untreated cells, a L1210 cells control, 0 plus 3AB. b M3 cells control, a plus 3AB... 

Neutral sucrose gradient sedimentation analysis under high salt conditions [3] shows (Fig. 1) that nucleoids from cells exposed to 100 fjM methotrexate for 1 h sediment more slowly than controls [ratio 0.856 0.04 (SD), n = 3]. If we accept that this reduction in sedimentation rate is due to DNA strand breaks this represents about 300 breaks per genome [4]. 

Since methotrexate diminishes tetrahydrofolate (FH4) levels it effectively inhibits not only thymidylate synthesis but that of purines and some amino acids also requiring FH4. In order to isolate the effect of the methotrexate to that on dihydrofolate reductase, and hence thymidylate synthesis, cells were grown with hypoxanthine (25 fjM) present also. Subsequent analysis revealed that nucleoids still sedimented more slowly than controls [ratio 0.879 0.055 (SD), n = 4]. 

During the DMSO-induced differentiation of HL-60 cells along the granulocytic lineage DNA, strand breaks are formed. This is reflected in the reduced sedimentation rate of the nucleoids during the first 24 h of treatment with DMSO, as compared to the sedimentation rate of nucleoids from parallel uninduced cultures (Fig. 6). At this time... 

Fig. 4. DNA strand break assay. DNA strand breaks were monitored by sedimentation of nucleoids through a 15-30% neutral sucrose gradient. Migration of nucleoids is expressed relative to migration of control (undamaged) nucleoids. A 24 h treatment with TG(0) control ( ) +SAB. B 30 min treatment with DMS. C 30 min treatment with DMS + SAB.

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