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O 6 -Methylguanine-DNA-methyltransferase (MGMT) is a key DNA repair enzyme involved in chemoresistance to DNA-alkylating anti-cancer drugs such as Temozolomide (TMZ) through direct repair of drug-induced O 6 -methylguanine residues in DNA. MGMT substrate analogues, such as O 6 -benzylguanine (BG), efficiently inactivate MGMT in vitro and in cells; however, these drugs failed to reach the clinic due to adverse side effects. Here, we designed hybrid drugs combining a BG residue covalently linked to a DNA-interacting moiety (6-chloro-2-methoxy-9-aminoacridine). Specifically, two series of hybrids, encompassing three compounds each, were obtained by varying the position of the attachment point of BG (N 9 of guanine vs. the benzyl group) and the length and nature of the linker. UV/vis absorption and fluorescence data indicate that all six hybrids adopt an intramolecularly stacked conformation in aqueous solutions in a wide range of temperatures. All hybrids interact with double-stranded DNA, as clearly evidenced by spectrophotometric titrations, without intercalation of the acridine ring and do not induce thermal stabilization of the duplex. All hybrids, as well as the reference DNA intercalator (6-chloro-2-methoxy-9-aminoacridine 8), irreversibly inhibit MGMT in vitro with variable efficiency, comparable to that of BG. In a multidrug-resistant glioblastoma cell line T98G, benzyl-linked hybrids 7a-c and the N 9 -linked hybrid 19b are moderately cytotoxic (GI 50  ≥ 15 μM after 96 h), while N 9 -linked hybrids 19a and 19c are strongly cytotoxic (GI 50  = 1-2 μM), similarly to acridine 8 (GI 50  = 0.6 μM). Among all compounds, hybrids 19a and 19c, similarly to BG, display synergic cytotoxic effect upon co-treatment with subtoxic doses of TMZ, with combination index (CI) values as low as 0.2-0.3. In agreement with in vitro results, compound 19a inactivates cellular MGMT but, unlike BG, does not induce significant levels of DNA damage, either alone or in combination with TMZ, as indicated by the results of γH2AX immunostaining experiments. Instead, and unlike BG, compound 19a alone induces significant apoptosis of T98G cells, which is not further increased in a combination with TMZ. These results indicate that molecular mechanisms underlying the cytotoxicity of 19a and its combination with TMZ are distinct from that of BG. The strongly synergic properties of this combination represent an interesting therapeutic opportunity in treating TMZ-resistant cancers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Some 60 years ago chemicals that intercalate between base pairs of duplex DNA were found to amplify frameshift mutagenesis. Surprisingly, the robust induction of frameshifts by intercalators still lacks a mechanistic model, leaving this classic phenomenon annoyingly intractable. A promising idea of asymmetric half-intercalation-stabilizing frameshift intermediates during DNA synthesis has never been developed into a model. Instead, researchers of frameshift mutagenesis embraced the powerful slipped-mispairing concept that unexpectedly struggled with the role of intercalators in frameshifting. It is proposed that the slipped mispairing and the half-intercalation ideas are two sides of the same coin. Further, existing findings are reviewed to test predictions of the combined "half-intercalation into the slipped-mispairing intermediate" model against accumulated knowledge. The existence of potential endogenous intercalators and the phenomenon of "DNA bookmarks" reveal ample possibilities for natural frameshift mutagenisis in the cell. From this alarming perspective, it is discussed how the cell could prevent genome deterioration from frameshift mutagenesis., (© 2019 WILEY Periodicals, Inc.)

Acridines are considered an important class of compounds due to their wide variety of biological activities. In this work, we synthesized four acridine derivatives (1-4) and evaluated their biological activity against the Plasmodium falciparum W2 line, as well as studied the interaction with ctDNA and HSA using spectroscopic techniques and molecular docking. The acridine derivative 2 (IC 50  = 0.90 ± 0.08 μM) was more effective against P. falciparum than primaquine (IC 50  = 1.70 ± 0.10 μM) and similar to amsacrine (IC 50  = 0.80 ± 0.10 μM). In the fluorescence and UV-vis assays, it was verified that the acridine derivatives interact with ctDNA and HSA leading to a non-fluorescent supramolecular complex formation. The non-covalent binding constants ranged from 2.09 to 7.76 × 10 3  M -1 , indicating moderate interaction with ctDNA. Through experiments with KI, fluorescence contact energy transfer and competition assays were possible to characterize the main non-covalent binding mode of the acridines evaluated with ctDNA as intercalation. The binding constants obtained showed a high linear correlation with the IC 50 values against the antimalarial activity, suggesting that DNA may be the main biological target of these molecules. Finally, HSA interaction studies were performed and all evaluated compounds bind to the site II of the protein. The less active compounds (1 and 3) presented the highest affinity to HSA, indicating that the interaction with carrier protein can affect the (bio)availability of these compounds to the biological target., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Acridone is highly fluorescent and stable against photodegradation, oxidation, and heat. It is also a small molecule with no charge, making it a promising fluorescent agent for use in a DNA probe. Thus, we have prepared 5'-terminal acridone-labeled DNAs by post-modification, and have examined their photophysical properties and their use as donors for a fluorescence resonance energy transfer (FRET) system in combination with a 3'-terminal dabcyl-tagged DNA as an acceptor, which can detect the target DNA by emission-quenching caused by FRET. The FRET with an acridone and dabcyl pair has been found to complement that with fluorescence and dabcyl and other fluorescence-quencher pairs. Significant amounts of quenching of the acridone emissions by guanine in the DNA were observed when guanine was close to acridone, which can be applied as a quencher-free probe for the detection of special sequence of DNA. The DNA bearing acridone at the C5 position of inner thymidine could discriminate the opposite T-T base mismatch, although enhancement of discrimination ability is needed for the practical use of SNP typing.

Detection of double stranded DNA and estimation of the AT content in DNA of unknown concentration could be achieved with a bis-acridinyl peptide carrying fluorescein, FKA.

The structure of the DNA binding site of the Nuclear single-stranded Binding Factor (NssBF), located in the long terminal repeat of the Drosophila 1731 retrotransposon, was investigated by melting temperature experiments, chemical probing and fluorescence measurements using a macrocyclic bis-acridine. The most probable structure of this element, named Bc, mainly involves two hairpins in equilibrium at pH 6.0 at low concentration. The hairpins differ in their apical loop size; 4 and 8 nt. The structural flexibility of Bc probably derives from the three consecutive CATA repeats complementary to the GTAT nucleotides of the palindrome. In contrast, the Bc complementary strand adopts a single hairpin. Since Bc is implicated in repression of transcription via binding of two specific factors, its structural flexibility could be associated with this process.

Triple helix formation usually requires an oligopyrimidine*oligopurine sequence in the target DNA. A triple helix is destabilized when the oligopyrimidine*oligopurine target contains one (or two) purine*pyrimidine base pair inversion(s). Such an imperfect target sequence can be recognized by a third strand oligonucleotide containing an internally incorporated acridine intercalator facing the inverted purine*pyrimidine base pair(s). The loss of triplex stability due to the mismatch is partially overcome. The stability of triplexes formed at perfect and imperfect target sequences was investigated by UV thermal denaturation experiments. The stabilization provided by an internally incorporated acridine third strand oligonucleotide depends on the sequences flanking the inverted base pair. For triplexes containing a single mismatch the highest stabilization is observed for an acridine or a propanediol tethered to an acridine on its 3'-side facing an inverted A*T base pair and for a cytosine with an acridine incorporated to its 3'-side or a guanine with an acridine at its 5'-side facing an inverted G*C base pair. Fluorescence studies provided evidence that the acridine was intercalated into the triplex. The target sequences containing a double base pair inversion which form very unstable triplexes can still be recognized by oligonucleotides provided they contain an appropriately incorporated acridine facing the double mismatch sites. Selectivity for an A*T base pair inversion was observed with an oligonucleotide containing an acridine incorporated at the mismatched site when this site is flanked by two T*A*T base triplets. These results show that the range of DNA base sequences available for triplex formation can be extended by using oligonucleotide intercalator conjugates.

Purine-rich (GA)- and (GT)-containing oligophosphorothioates were investigated for their triplex-forming potential on a 23 bp DNA duplex target. In our system, GA-containing oligophosphorothioates (23mer GA-PS) were capable of triplex formation with binding affinities lower than (GA)-containing oligophosphodiesters (23mer GA-PO). The orientation of the third strand 23mers GA-PS and GA-PO was antiparallel to the purine strand of the duplex DNA target. In contrast, (GT)-containing oligophosphorothioates (23mer GT-PS) did not support triplex formation in either orientation, whereas the 23mer GT-PO oligophosphodiester demonstrated triplex formation in the antiparallel orientation. GA-PS oligonucleotides, in contrast to GT-PS oligonucleotides, were capable of self-association, but these self-associated structures exhibited lower stabilities than those formed with GA-PO oligonucleotides, suggesting that homoduplex formation (previously described for the 23mer GA-PO sequence by Noonberg et al.) could not fully account for the decrease in triplex stability when phosphorothioate linkages were used. The 23mer GA-PS oligonucleotide was covalently linked via its 5'-end to an acridine derivative (23mer Acr-GA-PS). In the presence of potassium cations, this conjugate demonstrated triplex formation with higher binding affinity than the unmodified 23mer GA-PS oligonucleotide and even than the 23mer GA-PO oligonucleotide. A (GA)-containing oligophosphodiester with two phosphorothioate linkages at both the 5'- and 3'-ends exhibited similar binding affinity to duplex DNA compared with the unmodified GA-PO oligophosphodiester. This capped oligonucleotide was more resistant to nucleases than the GA-PO oligomer and thus represents a good alternative for ex vivo applications of (GA)-containing, triplex-forming oligonucleotides, allowing a higher binding affinity for its duplex target without rapid cellular degradation.

We have investigated the binding of short (10 base) acridine-linked triplex-forming oligonucleotides to the target sequence A6G6.C6T6 by DNase I footprinting. Specific binding is detected at low pH (< 6.0) for 5'-Acr-T5C5 and 5'-Acr-5BrU5(5Me)C5. The sequence T5C5, lacking the acridine modification, binds less strongly, though specific binding is still evident. 5'-Acr-T5C5 produces footprints at slightly lower concentrations than 5'-Acr-5BrU5(5Me)C5. All three oligonucleotides produce enhanced DNase I digestion at the 3'-end of the target purine strand, suggesting that there is a DNA structural change at the triplex-duplex boundary. Target sequences AnG4A and TAC3Tn, containing one and two triplex mismatches, show no interaction with the acridine-free oligonucleotide, but bind the acridine-linked oligonucleotides. In these secondary binding modes the third strand is positioned so that the mismatches are located at the 3'-end of the oligonucleotide. Mithramycin and distamycin, binding in the minor groove to GC- and AT-rich sequences respectively, abolish triple helix formation.

A study of the kinetics of renaturation of the DNP in the nuclei of human peripheral blood lymphocytes with the aid of modified Riglers method showed the absence of renaturation of the preparations preliminarily headed to 65 degrees C. Heating the preparations to higher temperatures (75, 85 and 100 degreesC) led to the appearance of characteristic renaturation profiles with the maximum at a two-minute incubation (direct measurement of the intensity of fluorescence of the nuclei of cells transfered into a cold citrate-salt solution, as a time function).

The steady-state prompt fluorescence, phosphorescence and delayed fluorescence spectra and triplet lifetimes of free proflavin and proflavin bound to native DNA and alternating poly d(A-IU) were obtained as a function of temperature in a buffer-glycerol solvent. The intensity of the proflavin E-type delayed fluorescence (DF) relative to both the phosphorescence (Ph) and the prompt fluorescence (F) was observed to increase with temperature, and plots of both ln (DF/Ph) and ln (DF/(F.tau T] as a function of 1/T were linear over a wide range of temperatures. Although the activation energies for the thermal repopulation of the proflavin excited singlet state from the triplet obtained from the slopes of these plots were essentially unchanged on binding, perturbations to the S1----T1 intersystem crossing rate constants extracted from the intercepts at infinite temperature were observed. The marked enhancement of the intersystem crossing that occurs with binding to the iodinated polynucleotide reflects an external heavy atom perturbation upon the intercalated dye which also induces a shortening in the triplet lifetime. With proflavin bound to DNA an enhancement to the S1----T1 intersystem crossing, though lesser in magnitude than for poly d(A-IU), is observed but with no change to the triplet lifetime. The well-studied fluorescence quenching of DNA-bound proflavin is a result of this increase in the intersystem crossing. It is proposed that these non-heavy atom enhancements in the intersystem crossing are due to distortions of the molecular plane of the bound proflavin molecule. In total these analyses provide a complete description of the excited state processes of the proflavin molecule and their variations with temperature.

Induction of peroxide free radicals (detected by Electron Paramagnetic Resonance at 77 K) due to the photodynamic activity of proflavine was measured on bacteriophage phi X174 DNA either single-stranded (ss) as isolated from the virion, or double-stranded supercoiled (RFI) as isolated from the infected bacteria. Comparison was made with calf thymus DNA photosensitization. In order to use equivalent DNA-proflavine complexes, binding of the dye to the three DNA's was first determined under those conditions of high ionic strength favourable to the photodynamic reaction. Free radical induction was maximal for definite amounts of bound proflavine (which varied depending upon the DNA substrate) and at an ionic strength value of 0.5. The level of the maximal reaction increased in the following order: from phi Xss DNA to calf thymus DNA and finally to phi XRFI DNA. The conformation of the proflavine-DNA complex was thus a determinant for the efficiency of the photodynamic process. The ionic strength effect could not be explained by the evolution of the proflavine triplet state in irradiated proflavine-calf thymus DNA complexes.

Actinomycin D specifically quenches the fluorescence of acridine orange and quinacrine bound to deoxyribonucleic acid in cytologic preparations, but does not change the fluorescence of these fluorochromes bound to RNA. The following fluorescence-cytochemical applications of techniques based on these findings can be suggested: (a) distinction between deoxyribonucleic acid and ribonucleic acid; (b) detection of double-stranded virus ribonucleic acid; (c) approximate estimation of the lengths of A-T sequences in deoxyribonucleic acid molecules.

Difference absorption spectra (complex-sum of the initial reagents) are obtained in the visible and longwave UV region for the system of actiflavine and DNA in a number of cases differing in initial and final degrees of DNA filling by the dye, in particular separately for two types of dye binding to DNA. For these binding types conventional absorption spectra are calculated. In the visible region for the first binding type ("strong" binding) red shift of the absorption band is observed; for the second type ("weak" binding) we observed splitting of the band, short wavelength component being highly prevailing, and hypochromism. In the UV region for both binding types the spectra changed in approximately similar way; a slight blue shift and a rather remarkable hypochromism are observed. It is shown that the dye brings the main contribution into the spectral changes in the UV region. If to take into account the spectral properties of molecular aggregates the data obtained are compatible with the intercalation model for "strong" binding and dye stacking on DNA for "weak" binding.

An oligodeoxythymidylate, oligo [d(T8)], was covalently linked to an acridine derivative via its 3' end and to EDTA via its 5' end. The octathymidylate was targeted to a single-stranded DNA fragment 27 nucleotides in length containing an octadeoxyadenylate sequence. In the presence of Fe(II) and a reducing agent (dithiothreitol) cleavage reactions were induced in the nucleotide sequence. The extent of the reaction was dependent on oligo concentration, salt concentration and temperature. Dissociation of the complexes at high temperature or low salt concentration abolished the site-specific cleavage reactions. Treatment of the reacted DNA with piperidine or piperidine-formiate strongly enhanced the yield of cleavage reactions demonstrating that damages were induced on nucleic acid bases by the EDTA-Fe complex covalently linked to the octathymidylate. At high salt concentration (1 M NaCl) or in the presence of spermine and ethylene-glycol a triple helix was formed involving the 27-mer DNA fragment and two oligo[d(T8)]. One of the oligo[d(T8)] was bound parallel and the other antiparallel to the oligo[d(A8)] complementary sequence. Cleavage reactions were induced on both sides of this oligo[d(A8)] target sequence. When a 27-mer duplex was used as a target the oligo[d(T8)] was bound in a parallel orientation with respect to the oligo[d(A8)]-containing strand in the major groove of the double helix. Cleavage reactions were induced on the oligo[d(A8)]-containing strand by the EDTA-Fe chelate attached to the 5' end of the oligo[d(T8)].

Anisotropy of torsional strength of the splitted electronic transition in the case of chromophore-chromophore interaction of dye molecules situated on the helical matrix was considered theoretically and as analytical expression for the value Rperpendicular/Rparallel was obtained. These theoretical results were compared with the experimental data obtained with DNA-proflavine, DNA-pyronine and DNA-acridine orange complexes oriented in multicappilar flow-cell. Studies of the optical effects (optical density and CD changes) due to orientation of these complexes showed that the acridine chromophores are not perpendicular with respect to the DNA axis (alpha D = 19--22 degrees). The DNA base pairs in complexes as assumed also are not perpendicular to the DNA axis, the inclination angle of their transition moments (for the band near 260 nm) being bigger than that of dye chromophores (24 degrees). These results indicate that under experimental conditions used by us no intercalation can be observed.

The crystal structure of a complex containing deoxy CpG and a terpyridine platinum compound (TPH) shows a DNA double helical fragment with TPH intercalated between two Watson-Crick GC base pairs. The DNA unwinding angle is 23 degrees and the pucker of the deoxyribose rings differ at the 3' and 5' ends.

Structures resembling nuclei but depleted of protein may be released by gently lysing cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids sediment in gradients containing intercalating agents in a manner characteristic of DNA that is intact, supercoiled and circular. The concentration of salt present during isolation of human nucleoids affects their protein content. When made in I-95 M NaCl they lack histones and most of the proteins characteristic of chromatin; in 1-0 M NaCl they contain variable amounts of histones. The effects of various treatments on nucleoid integrity were investigated.

The fading behavior of the 670 nm fluorescence emission band produced by unfixed rat mast cells stained with acridine orange (AO) has been found to be in excellent agreement with the behavior predicted by second order chemical kinetics. The reciprocal of fluorescence intensity plotted against time yields a straight line. When due account is taken of dye/cell ratio and the intensity of fluorescence-exciting radiation, Io (measured with the standard phosphor particle), the slope of this straight line is a constant, k'', which is independent of dye/cell ratio and Io. k'' differs from the second order photochemical rate constant by a constant factor. The fading of a given AO-biopolymer complex is described by a particular value of k''. Two values of k'' have been found for rat mast cell granules, indicating the presence of two different AO-biopolymer complexes. Fading of fluorescence may serve to identify particular intracellular biopolymers in individual cells even when present in a heterogeneous population.

This study examines certain aspects of the interaction of acridines with DNA. A comparative study of the methods available for the determination of the association constants (Kap) for compounds which interact with DNA has been pursued. A new equation which permits the spectrophotomeric determination of Kap has been derived. This equation can be applied to compounds which upon interaction with a polymer exhibit discrete absorption changes. Application of this equation to substituted acridines and tetrahydroacridines yields some preliminary information on the effect of ring substituents on the interaction of acridines with DNA. Low levels of DNA/dye ratios have been used in the studies reported herein.

In situ denaturation of nuclear deoxyribonucleic acid (DNA) is studied by use of acridine orange to differentially stain native versus denatured DNA, and a flow-through cytofluorometer for measurements of cell fluorescence. Thermal- or acid-induced DNA denaturation is markedly influenced by formaldehyde. Two mechanisms of the formaldehyde action are distinguished. If cells are exposed to the agent during heating, DNA denaturation is facilitated, most likely by the direct action of formaldehyde as a "passive" denaturing agent on DNA. If cells are pretreated with formaldehyde which is then removed, DNA resistance to denaturation increases, presumably due to chromatin cross-linking. It is believed that both effects occur simultaneously in conventional techniques employing formaldehyde to study DNA in situ, and that the extent of each varies with the temperature and cell type (chromatin condensation). Thus, profiles of DNA denaturation of cells heated with formaldehyde do not represent characteristics of DNA denaturation in situ; DNA denaturation under these conditions is modulated by the reactivity of chromatin components with formaldehyde rather than by DNA interactions with the macromolecules of nuclear mileu.

The fluorometric behaviour of cellular objects is influenced during excitation by two nearly independent phenomena: (1) by the photochemical reaction of the DNA/AF dye complex, and (2) by the energy transfer among several DNA/AF dye complexes. Both processes show a distinct temperature-dependent behaviour and can therefore be characterized by the analysis of the fluorescence spectra at different temperatures. All microfluorometric measurements were performed with a self-constructed cooling device. The cryostat permits measurements of the cellular fluorescence within a range of temperatures between 4 K and 300 K. The cooling unit operates in accordance with the 'Continuous Flow Principle' and allows the application of objectives up to a numerical aperture of 0.6.

Acid hydrolysed DNA of rat liver was stained with Schiff's reagent at pHs 1.7 or 3.0 followed by staining with acriflavine-SO2 at pH 2.0 as well as with acriflavine-SO2 followed by Schiff's reagent at pH 1.7 or 3.0. Nuclei stained with Schiff's reagent at pH 1.7 were brown-yellow and an analysis of their absorption characteristics revealed two peaks--one at 470 nm and the other at 570 nm. Although nuclei stained with Schiff's reagent at pH 3.0 followed by acriflavine-SO2 were deep magenta in colour, they also showed similar peaks of maximum absorption. Identical peaks were also seen when the sequence of staining was reversed. It is suggested that in the conventional Feulgen-type reactions only some of the DNA-aldehyde molecules are stained; the remaining molecules can be stained by sequential application of another Schiff or Schiff-type reagent such as acriflavine-SO2. The possible mechanism of staining in these cases has been discussed.

The interaction between acridine orange (AO) and diluted and concentrated solutions of DNA, DNP systems and chromatin suspension at the physiologic ionic strength was investigated. The effect of AO on DNP systems was also investigated. It was shown that highest possible number of AO molecules bound to DNA made up 70% of the total number of nucleotides. The model of AO binding to DNA is proposed and used for calculation of constants of stronger and weaker AO-binding capacities equal to 6-10(6) M-1 and 1,7-10(5) M-1, respectively. The AO-DNA binding constants in DNP-complex are five as low. The primary number of binding sites in chromatin suspension made up 10% of the corresponding sites in DNA and increased as AO was adsorbed. AO induced the supercontraction of oriented DNP systems at the physiologic ionic strength and the appearance of the low-temperature melting hump.

In the presence of sulfhydryl compounds, an anticancer drug 1-nitro-9-(3-N,N-dimethylaminopropylamino) acridine (nitracrine, Ledakrin) forms irreversible complexes of decreased template activity with DNA. Stoichiometry of the complexes was estimated using the drug labelled with 14C in the acridine ring or in the propyl chain and with 3H in the acridine ring. Up to 50 irreversibly bound 14C-nitracrine molecules were found per 10(3) nucleotides of calf thymus DNA in the presence of dithiothreitol (DTT). Considerably lower binding observed using 3H-labelled drug, particularly when the complexes were formed in the presence of mercaptoethanol (ME) indicates that substitution of tritium atoms occurred during the reaction. Relationship between stoichiometry and template activity in RNA synthesis in vitro system of the complexes was estimated in the paper.

As a first step in the synthesis and the study of DNA polyintercalating drugs, dimers of acridines have been prepared. Their DNA binding properties have been studied. It has been determined that when the chain separating the two aromatic rings is longer than a criticål distance, bisintercalation is actually observed and that the DNA binding affinity becomes quite large (greater than 10(8)-10(9) M-1). It is shown also that the optical characteristics of these molecules are dependent on the sequences of DNA. The fluorescence intensity of one of these dimers when bound to DNA varies as the fourth power of its A+T content. This derivative could be used as a fluorescent probe of DNA sequence.

Several parameters of stimulation of individual lymphocytes are measured simultaneously by flow-cytofluorometry after differential staining of cellular DNA and RNA with the metachromatic fluorescent dye acridine orange. The method provides a means of analyzing the progression of stimulated cells through the cell cycle (G1, S and G2 + M), in addition to measuring the accumulation of RNA per cell; the RNA parameter is useful in distinguishing G1 from G0 cells. The multiparameter analysis may be of unique value in discriminating cases where the transcriptional and proliferative responses of lymphocytes to stimulants are not correlated.

Cells in mitosis may be distinguished from interphase cells based on difference in chromatin structure as revealed by two different methods of staining with acridine orange. In the first method, cells are heated and then stained at neutral pH; the difference in stainability between mitotic and interphase cells reflects the difference in the extent of deoxyribonucleic acid denatured by heat in these cells. At a given temperature the deoxyribonucleic acid of the mitotic cell appears to be more extensively denatured than that of the interphase cell. In the second method, cells are treated with buffer at pH 1.5 (1.3 to 1.9) and then stained at pH 2.6 (2.3 to 2.9). The mechanisms involved in the differential stainability of interphase versus mitotic cells at that low pH are currently under investigation. In both methods, in addition to enumerating cells in mitosis, it is possible to quantitate cells in G1, S and G2 phases of the cell cycle.