Use of the method of fast atom bombardment mass spectrometry for identification of products of the interaction of thiophosphamide with DNA

Products of interaction between DNA and an antitumour drug N, N', N'-triethylenethiophosphoramide (thiotepa) have been observed for the first time by the fast atom bombardment mass spectrometry. The sites of alkylation are detected as N7 (Gua) and N3 (Ade), and yields of the products are evaluated.     

Direct observation of the alkylation products of deoxyguanosine and DNA by fast atom bombardment mass spectrometry.

By the methods of fast atom bombardment (FAB) mass spectrometry, thin-layer chromatography and ultraviolet absorption spectroscopy adducts have been studied which are formed by an antitumour alkylating drug thiotepa both in a model system, containing only deoxyguanosine (dGuo), and in DNA. Analysis of the model reaction mixture (dGuo + thiotepa) by FAB mass spectrometry permitted observation of adducts dGuo thiotepa, 2dGuo thiotepa, and also the products of their further modification in solution, which occurs by hydrolysis of the glycosidic bond and also by opening of the imidazole ring. In the case of DNA FAB mass spectrometry made it possible to characterize adducts of thiotepa with guanosine (Gua) and adenosine (Ade) without their preliminary purification. The site of alkylation of Gua in both dGuo and DNA is N7, and that of Ade in DNA is N3. The application of the results to the study of the molecular mechanism of the antitumour action of thiotepa is discussed.     

Alkylation of nucleic acid components with ethylenimine and its derivatives. IV. Alkylation of homopolynucleotides and DNA]

Alkylation of homopolynucleotides and DNA by thio TEPA and monoaziridine diethyl phosphate was studied. The modification affected nucleic bases and terminal phosphate groups but not internucleotide phosphate groups. It was shown that the main center of modification in poly(A) was the N1 atom, whereas the products of N6- and N3-alkylations were formed in smaller amounts. In poly(G), the alkylation proceeded predominantly at the N7 and, insignificantly, at the N1 atom of guanine; the pyrimidine N3 atom is alkylated poorly in poly(C) and even worse in poly(U). In the case of DNA, the major alkylated sites are the guanine N7 and the adenine N3; this results in DNA denaturation and the subsequent formation of products modified at N1 and N6 of adenine, N1 of guanine, and N3 of cytosine. An increase in the pH and ionic strength of the solution as well as the DNA denaturation decrease the reaction rate, whereas ultrasonic fragmentation enhances it. Upon alkylation, melting temperatures decrease, CD and UV spectra change, and DNA luminescence appears. To separate the reaction mixtures and identify the DNA alkylation products, chemical hydrolysis, ion-exchange and reverse-phase HPLC, and UV spectroscopy were used.     

The influence of some alkylating agents on the structure of DNA in vitro

The influence of the industrially used mutagenic agents β-propiolactone (BPL), propylene oxide (PO) and butylene oxide (BO) on the structure of DNA in vitro was studied. The heat denaturation of DNA and its reversibility were used as a criterion of the structural change in the DNA molecule. The rate constants for the reaction of the different compounds with DNA were determined. The effects were correlated with the degree of alkylation. Butylene oxide and propylene oxide caused a decrease of the reversibility of the heat denaturation at a degree of alkylation at which the melting temperature was only slightly decreased. β-propiolactone had no influence on the reversibility, but decreased the melting temperature of DNA as a function of the degree of alkylation.     

Studies on interaction between poly(L-lysine58, L-phenylalanine42) and deoxyribonucleic acids.

A random copolymer of 58% L-lysine and 42% L-phenylalanine, poly(Lys58Phe42), was used as a model protein for studying the role of phenylalanine residues in protein-DNA interaction. Complexes between this copolypeptide and DNA, made by direct mixing, were studied by absorbance, circular dichroism (CD), fluorescence, and thermal denaturation. Complex formation results in an increase in absorbance, and an enhancement, red-shift, and broadening of phenylalanine fluorescence. The fluorescence enhancement is opposite to the quenching observed when a tyrosine copolypeptide is bound to DNA (R. M. Santella and H.J. Li (1974), Biopolymers 13, 1909). The positive CD band of DNA near 275 nm is reduced and red-shifted by the binding of the phenylalanine copolypeptide to a greater extent than by the tyrosine copolypeptide. Thermal denaturation of the complexes in 2.5 times 10(-4) M EDTA (pH 8.0) shows three characteristic melting bands. For complexes with calf thymus DNA, free base pairs melt at Tm,I (47-49 degrees) and copolypeptide-bound base pairs show two melting bands (Tm,II at 73-75 degrees, and Tm,III at 88 -90 degrees). Similar thermal denaturation results have been observed for complexes with Micrococcus luteus DNA. The fluorecence intensity of the complexes is greatly increased when the temperature is raised to the Tm,II region. In addition to fluorescence measurements, the effects of increasing temperature on absorption and CD spectra of the complexes were also studied. Stacking interaction between the phenylalanine chromophore and DNA bases, either partial or full intercalation, is implicated by the experimental results. Several mechanisms are proposed to describe the reaction between the copolypeptide and DNA, and thermal denaturation of the complex.     

Association constants for the interaction of double-stranded and single-stranded DNA with spermine, spermidine, putrescine, diaminopropane, N1- and N8-acetylspermidine, and magnesium: determination from analysis of the broadening of thermal denaturation curves

The effect of Mg2+, putrescine, diaminopropane, N1-acetylspermidine, N8-acetylspermidine, spermidine, and spermine on the thermal denaturation of calf thymus DNA was investigated. As in a previous study with magnesium [W.F. Dove and N. Davidson, (1962) J. Mol. Biol. 5, 467-478], these ligands were found to raise the thermal denaturation temperature of the DNA and to broaden the thermal denaturation curve dramatically at the point where 10 to 20% of the DNA charge had been neutralized. At higher levels of charge neutralization the curves became sharper again. This behavior was due to differential binding of the ligands to single- and double-stranded DNA. The broadening was used to determine the ratio of the association constants of each ligand to the two forms of DNA using either an independent sites model of binding or an excluded sites model. The results show that the primary mode of binding of the ligands to DNA is electrostatic but that important secondary, nonelectrostatic, effects are also present.     

Effects of denaturation with HCl on the immunological staining of bromodeoxyuridine incorporated into DNA

Immunochemical procedures for detection of BrdUrd incorporated into DNA require a denaturation step of DNA. Denaturation with HCl is widely used for flow cytometric analysis of the cell cycle and for histological preparations. This brief communication describes an attempt to standardize a denaturation procedure with HCl. Various denaturation conditions at 20 degrees C were examined for human promyelocytic leukemia cells (HL-60 cells) fixed in ethanol. After denaturation of DNA, the cells were stained by an indirect immunofluorescence method using a commercially available monoclonal anti-BrdUrd antibody or by propidium iodide. The relative fluorescence intensities of stained BrdUrd and double-stranded DNA were altered reciprocally by changing HCl concentration and/or denaturation time. Treatment with 4N HCl for 10-20 min at 20 degrees C allowed denaturation of more than 80% of DNA and the maximum BrdUrd-linked immunofluorescence. Under this condition, the coefficient of variation of the DNA histograms remained relatively small.     

Denaturation of covalently closed circular DNA. Kinetics, comparison of several DNAs, mechanism and ionic effects

The rates of the alkaline denaturation of the covalently closed, circular DNAs (form I) of the replicative forms (RF) of phages G4, phi X174, and fd, and of plasmid pBR322 and phage PM2 have been measured at 0 degrees C and some at higher temperatures. These rates are orders of magnitude slower than the denaturation of linear DNA because of the increased stability of the helix to deprotonation that results from the accumulating positive superhelicity during denaturation. Denaturation reactions were initiated by rapid, infrasonic mixing (Camien, M.N., and Warner, R.C. (1984) Anal. Biochem. 138, 329-334), and their progress was measured by analytical ultracentrifugal analysis for the amounts of form I and denatured (Id) DNA after neutralization of the alkaline reaction. The comparative rates of the five DNAs varied over a wide range; the fastest, G4-RF, denatured at 500-fold the rate of the slowest, fd-RF. The differences are accounted for by the interaction of positive superhelicity with the sequence-dependent regions of relative helix stability in the various DNAs. Renaturation rates of Id DNAs varied similarly for Ids prepared at 0 degrees C, but only a few-fold for Ids prepared at 50 degrees C. The rate of denaturation of G4-RF was determined over a wide range of NaOH and NaCl concentrations at 0 degrees C, and the pHm was determined as a function of ionic strength and temperature. The effects of ionic strength have been analyzed in an application of the Manning ion condensation-screening theory (Manning, G.S. (1978) Q. Rev. Biophys. 11, 179-246) which is shown to account for the large destablizing effect of salts on the helix. The pH region of transition at 50 degrees C from renaturation to denaturation was examined, and it was found that the maximum rate of renaturation occurred at a pH about 0.05 units below the pHm.     

DNA determinants important in sequence recognition by Eco RI endonuclease

Alkylation interference and protection methods (Siebenlist, U., and Gilbert, W., (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 122-126) have been utilized to deduce potential DNA contacts involved in specific complex formation between Eco RI endonuclease and its recognition sequence. The endonuclease protected the N7 position (major groove) of the dG and the N3 position (minor groove) of both dA residues within the Eco RI sequence against alkylation by dimethylsulfate, d(GpApApTpTpC), suggesting the presence of poly-peptide in both grooves in the vicinity of affected nitrogens. Results of methylation interference analysis suggest that the N7 of the Eco RI site dG and the N3 of the central dA, d(GpApApTpTpC), are utilized as contacts by the enzyme. The failure to observe interference upon methylation of the 5'-penultimate dA within the sequence implies that the endonuclease does not bond to the N3 of this residue, despite the fact that it is protected against alkylation by the protein. Ethylation interference patterns suggest four major phosphate contacts between endonuclease and each DNA strand. Two of these phosphates are 5'-external to the Eco RI sequence, d(pNpGpApApTpTpC), suggesting involvement of outside phosphates in electrostatic interactions. Moreover, alkylation protection and interference effects on the two DNA strands display perfect 2-fold symmetry. Thus, the endonuclease interacts with a minimum of 10 nucleotide pairs to yield a DNA-protein complex characterized by elements of symmetry. In contrast, specific alkylation effects were not observed in comparable experiments with the endonuclease and a DNA which had been previously methylated by the Eco RI modification enzyme.     

Studies related to antitumor antibiotics. Part V. Reactions of mitomycin C with DNA examined by ethidium fluorescence assay.

The cytotoxic action of the antitumor antibiotic mitomycin C occurs primarily at the level of DNA. Using highly sensitive fluorescence assays which depend on the enhancement of ethidium fluorescence only when it intercalates duplex regions of DNA, three aspects of mitomycin C action on DNA have been studied: (a) cross-linking events, (b) alkylation without necessarily cross-linking, and (c) strand breakage. Cross-linking of DNA is determined by the return of fluorescence after a heat denaturation step at alkaline pH's. Under these conditions denatured DNA gives no fluorescence. The cross-linking was independently confirmed by S1-endonuclease (EC 3.1.4.-) digestion. At relatively high concentrations of mitomycin the suppression of ethidium fluorescence enhancement was shown not to be due to depurination but rather to alkylation, as a result of losses in potential intercalation sites. A linear relationship exists between binding ratio for mitomycin and loss of fluorescence. The proportional decrease in fluorescence with pH strongly suggests that the alkylation is due to the aziridine moiety of the antibiotic under these conditions. A parallel increase in the rate and overall efficiency of covalent cross-linking of DNA with lower pH suggests that the cross-linking event, to which the primary cytotoxic action has been linked, occurs sequentially with alkylation by aziridine and then by carbamate. Mitomycin C, reduced chemically, was shown to induce single strand cleavage as well as monoaklylation and covalent cross-linking in PM2 covalently closed circular DNA. The inhibition of this cleavage by superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6), and by free radical scavengers suggests that the degradation of DNA observed to accompany the cytotoxic action of mitomycin C is largely due to the free radical O2. In contrast to the behavior of the antibiotic streptonigrin, mitomycin C does not inactivate the protective enzymes superoxide dismutase or catalase. Lastly, mitomycin C is able to cross-link DNA in the absence of reduction at pH 4. This is consistent with the postulated cross-linking mechansims.     

Adenine N3 is a main alkylation site of styrene oxide in double-stranded DNA

Styrene 7,8-oxide (SO), a major metabolite of styrene, is classified as a probable human carcinogen. In the present work, salmon testis DNA was reacted with SO and the alkylation products were analysed after sequential depurination in neutral or acidic conditions followed by HPLC separation and UV-detection. A novel finding was that the N-3 position of adenine was the next most reactive alkylation site in double-stranded DNA, comprising 4% of the total alkylation, as compared to alkylation at the N-7 position of guanine, 93% of the total alkylation. Both alpha- and beta-products of SO were formed at these two sites. Other modified sites were N2-guanine (1.5%, alpha-isomer), 1-adenine (0.4%, both isomers) and N6-adenine (0.7%, both isomers) as well as 1-hypoxanthine (0.1%, alpha-isomer), formed by deamination of the corresponding 1-adenine adduct. The results indicated that in double-stranded DNA N-7 of guanine and N-3 of adenine account for 97% of alkylation by SO. However, these abundant adducts are not stable, the half-life of depurination in DNA for 3-substituted adenines being approximately 10 and approximately 20 h, for alpha- and beta-isomers, respectively, and 51 h for both isomers of 7-substituted guanines.     

A differential scanning calorimetry study of tetracycline repressor.

Tetracycline repressor (TetR), which constitutes the most common mechanism of bacterial resistance to an antibiotic, is a homodimeric protein composed of two identical subunits, each of which contains a domain possessing a helix-turn-helix motif and a domain responsible for binding tetracycline. Binding of tetracycline in the protein pocket is accompanied by conformational changes in TetR, which abolish the specific interaction between the protein and DNA. Differential scanning calorimetry (DSC) and CD measurements, performed at pH 8.0, were used to observe the thermal denaturation of TetR in the absence and presence of tetracycline. The DSC results show that, in the absence of tetracycline, the thermally induced transitions of TetR can be described as an irreversible process, strongly dependent on scan rate and indicating that the protein denaturation is under kinetic control described by the simple kinetic scheme: N(2)--->D(2), where k is a first-order kinetic constant, N is the native state, and D is the denatured state. On the other hand, analysis of the scan rate effect on the transitions of TetR in the presence of tetracycline shows that thermal unfolding of the protein can be described by the two-state model: N(2)<--->U(2)--->D. In the proposed model, TetR in the presence of tetracycline undergoes co-operative unfolding, characterized by an enthalpy change (DeltaH(cal) = 1067 kJ x mol(-1)) and an entropy change (DeltaS = 3.1 kJ x mol(-1)).     

Detection of 5-bromodeoxyuridine (BrdUrd) incorporation by monoclonal antibodies: role of the DNA denaturation step

Immunochemical detection of cells that incorporate 5-bromodeoxyuridine (BrdUrd) requires prior denaturation of DNA in situ to make BrdUrd binding sites accessible to the antibodies. A technique is described in which the DNA denaturation step is facilitated by a) prior dissociation of histones from DNA and b) the use of low ionic strength buffer in which the cells are suspended during heating. Dissociation of histones is achieved by cell treatment with 0.08N HCl at 0 degree C, which a) increases accessibility of DNA to propidium iodide (and following the denaturation to the antibodies); b) lowers stability of DNA to thermal denaturation; c) decreases differences between various cell types due to variability in chromatin structure; and d) ensures more complete DNA denaturation. Cell heating (80-95 degrees C) at low ionic strength (1 mM Na+) eliminates the need for formamide and results in extensive and rapid DNA denaturation. The method was applied in Friend leukemia, L1210 and HL-60 cell lines, and to bone marrow, experimental animal tumor and primary human tumor cells.     

Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards

Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increased ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specific chemical cleavage technique for DNA sequencing. For L-Pam and UM, increased ionic strength and the cationic DNA affinity binders dose dependently inhibited the alkylation. QM alkylation was less inhibited by salt (100 mM NaCl), ethidium (10 microM), and spermine (10 microM). Distamycin A and netropsin (100 microM) gave an enhancement of overall QM alkylation. More interestingly, the pattern of guanine N7-alkylation was qualitatively altered by ethidium bromide, distamycin A, and netropsin. The result differed with both the nitrogen mustard (L-Pam less than UM less than QM) and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove.     

Transfection of Escherichia coli Spheroplasts II. Relative Infectivity of Native, Denatured, and Renatured Lambda, T7, T5, T4, and P22 Bacteriophage DNAs

The change of infectivity of phage DNAs after heat and alkali denaturation (and renaturation) was measured. T7 phage DNA infectivity increased 4- to 20-fold after denaturation and decreased to the native level after renaturation. Both the heavy and the light single strand of T7 phage DNA were about five times as infective as native T7 DNA. T4 and P22 phage DNA infectivity increased 4- to 20-fold after denaturation and increased another 10- to 20-fold after renaturation. These data, combined with other authors' results on the relative infectivity of various forms of phiX174 and lambda DNAs give the following consistent pattern of relative infectivity. Covalently closed circular double-stranded DNA, nicked circular double-stranded DNA, and double-stranded DNA with cohesive ends are all equally infective and also most highly infectious for Escherichia coli lysozyme-EDTA spheroplasts; linear or circular single-stranded DNAs are about 1/5 to 1/20 as infective; double-stranded DNAs are only 1/100 as infective. Two exceptions to this pattern were noted: lambda phage DNA lost more than 99% of its infectivity after alkaline denaturation; this infectivity could be fully recovered after renaturation. This behavior can be explained by the special role of the cohesive ends of the phage DNA. T5 phage DNA sometimes showed a transient increase in infectivity at temperatures below the completion of the hyperchròmic shift; at higher temperatures, the infectivity was completely destroyed. T5 DNA denatured in alkali lost more than 99.9% of its infectivity; upon renaturation, infectivity was sometimes recovered. This behavior is interpreted in terms of the model of T5 phage DNA structure proposed by Bujard (1969). The results of the denaturation and renaturation experiments show higher efficiencies of transfection for the following phage DNAs (free of single-strand breaks): T4 renatured DNA at 10(-3) instead of 10(-5) for native DNA; renatured P22 DNA at 3 x 10(-7) instead of 3 x 10(-9) for native DNA; and denatured T7 DNA at 3 x 10(-6) instead of 3 x 10(-7) for native DNA.     

Effect of different denaturing agents on the detectability of specific DNA sequences of various base compositions by in situ hybridisation

In situ hybridisation of certain AT rich and GC rich satellite DNA complementary RNAs (cRNAs) to their homologous chromosomes at their respective optimal rate temperatures (T_OPTS) after denaturation with various reagents (0.2 N HCl, 0.07 N NaOH, 90% formamide and heat) led to the following conclusions. — Heat denaturation of chromosomal DNA in 0.1×SSC at 100° C gives significantly higher grain counts regardless of DNA base composition, HCl denaturation discriminates markedly against GC rich DNA. Chromosome morphology is best preserved after HCl and heat denaturation.     

Unwinding of origin-specific structures by human replication protein A occurs in a two-step process.

The simian virus 40 (SV40) large tumor antigen(T antigen) has been shown to induce the melting of 8 bp within the SV40 origin of replication. We found previously that a 'pseudo-origin' DNA molecule (PO-8) containing a central 8 nt single-stranded DNA (ssDNA) bubble was efficiently bound and denatured by human replication protein A (hRPA). To understand the mechanism by which hRPA denatures these pseudo-origin molecules, as well as the role that hRPA plays during the initiation of SV40 DNA replication, we characterized the key parameters for the pseudo-origin binding and denaturation reactions. The dissociation constant of hRPA binding to PO-8 was observed to be 7.7 x 10(-7) M, compared to 9.0 x 10(-8) M for binding to an identical length ssDNA under the same reaction conditions. The binding and denaturation of PO-8 occurred with different kinetics with the rate of binding determined to be approximately 4-fold greater than the rate of denaturation. Although hRPA binding to PO-8 was relatively temperature independent, an increase in incubation temperature from 4 to 37 degreesC stimulated denaturation nearly 4-fold. At 37 degreesC, denaturation occurred on approximately 1/3 of those substrate molecules bound by hRPA, showing that hRPA can bind the pseudo-origin substrate without causing its complete denaturation. Tests of other single-stranded DNA-binding proteins (SSBs) over a range of SSB concentrations revealed that the ability of the SSBs to bind the pseudo-origin substrate, rather than denature the substrate, correlated best with the known ability of these SSBs to support the T antigen-dependent SV40 origin-unwinding activity. Our data indicate that hRPA first binds the DNA substrate using a combination of contacts with the ssDNA bubble and duplex DNA flanks and then, on only a fraction of the bound substrate molecules, denatures the DNA substrate.     

Chromosomal DNA denaturation and reassociation in situ.

The degree of chromosomal DNA (cDNA) denaturation and renaturation on polytene chromosomes has been measured by UV microspectrophotometry. Also DNA losses occurring upon denaturation have been quantified by Feulgen, gallocyanin-chromalum and UV. It has been observed that denaturation in alkali (0.07 N NaOH at room temperature) and formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C removes about 30% of the DNA. Low DNA loss occurs upon denaturation in HCl (0.24 M) at room temperature and 60% formamide: 2 × 10^?4 M EDTA (pH 8) at 55 °C. The presence of 4% formaldehyde in the denaturation buffer prevents DNA loss. After denaturation of chromosomes in 0.1 × SSC containing 4% formaldehyde at 100 °C for 30 sec, an hyperchromicity of 39 °C is observed. The denaturation efficiency varies with the denaturation treatment. The percentage reassociation was measured from the difference in the UV absorption of renatured chromosomes and that of denatured chromosomes from the same set. It seems that in our conditions DNA:DNA reassociation does not occur. The efficiency of hybridization is proportional to the denaturation extent of the DNA. However, the entire fraction of DNA which has been denatured is not available for hybridization.     

DNA-directed alkylating ligands as potential antitumor agents: sequence specificity of alkylation by intercalating aniline mustards

The sequence preferences for alkylation of a series of novel parasubstituted aniline mustards linked to the DNA-intercalating chromophore 9-aminoacridine by an alkyl chain of variable length were studied by using procedures analogous to Maxam-Gilbert reactions. The compounds alkylate DNA at both guanine and adenine sites. For mustards linked to the acridine by a short alkyl chain through a para O- or S-link group, 5'-GT sequences are the most preferred sites at which N7-guanine alkylation occurs. For analogues with longer chain lengths, the preference of 5'-GT sequences diminishes in favor of N7-adenine alkylation at the complementary 5'-AC sequence. Magnesium ions are shown to selectively inhibit alkylation at the N7 of adenine (in the major groove) by these compounds but not the alkylation at the N3 of adenine (in the minor groove) by the antitumor antibiotic CC-1065. Effects of chromophore variation were also studied by using aniline mustards linked to quinazoline and sterically hindered tert-butyl-9-aminoacridine chromophores. The results demonstrate that in this series of DNA-directed mustards the noncovalent interactions of the carrier chromophores with DNA significantly modify the sequence selectivity of alkylation by the mustard. Relationships between the DNA alkylation patterns of these compounds and their biological activities are discussed.     

Resistance to DNA denaturation in irradiated Chinese hamster V79 fibroblasts is linked to cell shape

Exponentially growing Chinese hamster V79-171b lung fibroblasts seeded at high density on plastic (approximately 7 x 10(3) cells/cm2) flatten, elongate, and produce significant amounts of extracellular fibronectin. When lysed in weak alkali/high salt, the rate of DNA denaturation following exposure to ionizing radiation is exponential. Conversely, cells plated at low density (approximately 7 x 10(2) cells/cm2) on plastic are more rounded 24 h later, produce little extracellular fibronectin, and display unusual DNA denaturation kinetics after X-irradiation. DNA in these cells resists denaturation, as though "constraints" to DNA unwinding have developed. Cell doubling time and distribution of cells in the growth cycle are identical for both high and low density cultures as is cell survival in response to radiation damage. The connection between DNA conformation and cell shape was examined further in low density cultures grown in conditioned medium. Under these conditions, cells at low density were able to elongate, and DNA denaturation of low density cultures was identical to that of high density cultures. Conversely, cytochalasin D, which interferes with actin polymerization causing cells to "round up" and release fibronectin, allowed development of constraints in high density cultures. These results suggest that DNA conformation is sensitive to changes in cell shape which result when cells are grown in different environments. However, these changes in DNA conformation detected by the DNA unwinding assay do not appear to play a direct role in radiation-induced cell killing.