Cytometry of the cell cycle: Cycling through history

For young investigators who presently enter their scientific pursuit focused on cell cycle and have flow cytometry (FCM) at their disposal, it may be difficult to visualize the hardship of experimental procedures during the precytometry era. Autoradiography (1) was then the predominant method for cell cycle studies. It was a cumbersome and time-consuming methodology. The radioisotope-labeled cells deposited on microscope slides had to be fixed and covered with photographic emulsion in nearly total darkness. This was particularly tricky when using the "stripping film" approach, and required the preparer to either be on a carrot diet or to consume large quantities of vitamin A to enhance his or her night vision. After careful air-drying, the autoradiographs had to be left in light-proof boxes for several days' and sometimes weeks' exposure. Then, again in the dark, the autoradiographs had to be processed through developer, rinse, and fixer, followed by drying. Subsequently, the cells had to be counterstained through the emulsion (which also was tricky, because the emulsion had a tendency to detach, ruining the specimen) and mounted under a coverslip. Quantitative analysis of autoradiographs was painful as well. One had to identify labeled cells located below the silver grains of emulsion by microscopy, and score by eye the labeling index (LI) by counting hundreds of cells per each sample. Sometimes it was necessary to count individual silver grains, to estimate the intensity of cell labeling. Such analysis could take a long time, leaving the investigator with painful eyes and phantom images of the silver grains residing in his or her retina for hours. Attempts to develop semiautomatic or automatic screening of autoradiographs based on backward light scattering by silver grains of emulsion were generally unsuccessful (1). Despite the hardship, important discoveries were made, and numerous autoradiographic techniques, designed to assess the cell cycle and kinetics of cell proliferation, were developed. In fact, the evidence that DNA replication is discontinuous during the cycle, occurring within the distinct time interval during the interphase, was obtained by autoradiography (2, 3). It was observed that the radioisotope (32P or 3H)-labeled DNA precursor thymidine was incorporated into nuclei by a fraction of the interphase cells only, leaving many cells with unlabeled nuclei. This finding provided the foundation for subdivision of the cell cycle into four major phases: pre-DNA synthetic interphase or postmitotic gap (G1), DNA synthesis phase (S), postsynthetic interphase or premitotic gap (G2), and mitosis (M). Perhaps the most elegant technique to measure kinetics of cell progression through the cycle was based on pulse-labeling cells with 3H-thymidine, followed by analysis of the percentage of labeled mitotic cells (3, 4). Analysis of the kinetics of progression of the cohort of cells labeled during the short pulse in S phase through the narrow time-window of the M phase provided an accurate estimate of the duration of each phase of the cycle and of the whole cell cycle (Tc) (4). In vitro and in vivo applications of 3H-thymidine autoradiography yielded a wealth of information about cell cycle and the kinetics of cell proliferation of several normal and cancer cell models (4-6). Microspectrophotometry and microfluorometry were also applied in studies of the cell cycle, as techniques complementary to autoradiography. They were used to measure the content of DNA, RNA, and protein in individual cells. However, only few laboratories could afford such instruments, which were then generally homemade. Their development and maintenance required a significant investment and close collaboration of biologists with optical and mechanic engineers. During 1967–1968, one of us (ZD) had an opportunity to use these instruments at the Institute for Medical Cell Research and Genetics at the Karolinska Institute in Stockholm. Directed by Torbjorn Caspersson, the "grandfather" of cytometry (7), this laboratory had the most advanced microspectrophotometers and microfluorometers at the time, and was the Mecca for scientists from all over the world who were seeking a possibility to quantify DNA, RNA, or total protein in individual cells. Reservations to use the microspectrophotometer had to be made weeks ahead, as there was a long waiting line of investigators eager to measure cells. After some experience was gained, approximately 40 cells could be measured per hour. This number was then considered be adequate for statistical analyses in most publications. Needless to say, the cell analysis had to be biased by prior visual selection of the subsequently measured cell. As one can imagine, the possibility of rapid and accurate measurements of cell constituents offered by FCM (8-10), when compared with the pains of pre-FCM era, made the cell cycle investigators extremely joyous. The speed of cell analysis by FCM was four to five orders of magnitude greater than slide-based microspectrophotometry. Thousands of cells per sample could be measured instead of only a few cells. The cell selection was unbiased and data analysis was rapid and semiautomatic. Instead of waiting weeks for results of the experiment, e.g., when applying autoradiography, FCM yielded the data almost instantaneously. Given the above, most researchers studying the cell cycle instantly become devotees of FCM. Research progress in cell biology, particularly in the cell cycle field, was dramatically accelerated. The Cell Kinetics Society, which later merged with the Cell Cycle Society to form the Cell Proliferation Society, has become the forum for FCM; most of their members were also members of the Society for Analytical Cytology (SAC; the precursor of ISAC). Table 1 lists mileposts in the development of FCM methods to probe the cell cycle. Because cellular DNA content alone yields information on cell position in G0/1 versus S versus G2/M, a variety of methods were initially developed to measure this cell attribute (Fig. 1). Historical progress in developing the DNA content using FCM methods was associated with the introduction of new fluorochromes and modifications of the cell staining procedures, as outlined below. Composite of DNA content histogram obtained from CHO cell populations in the exponential growth phase, or synchronized in various phases of the cell cycle, as shown. Cells were stained by the acriflavine-Feulgen procedure (11). In early attempts, the Feulgen fluorescent method, utilizing either auramine O (11), or the improved procedure, employing acriflavine (12), was introduced to FCM. As cumbersome and time consuming as the Feulgen procedure was in actual practice, it proved to be of significant value for validating the early applications of FCM. An article appearing in Science (10) in 1969 showed a good quality DNA histogram of auramine O Feulgen-stained CHO cells. The coefficient of variation (CV) of the mean fluorescence of G1 cells reported by these authors (6.4%) was acceptable for that period, and the fluorescence intensity G2+M to G1 peak ratio was 1.97, close to the expected value of 2.00. Cell volume distributions were also measured and were different from those for the Feulgen-DNA content. These data countered the early criticisms of the technique, which implied that the procedure was actually measuring the nuclear or the cell volume. To further substantiate credibility of the flow cytometric technique, cells were synchronized in the various phases of the cell cycle and analyzed by flow microfluorometry (FMF), as it was often called at the time. The composite in Figure 1 shows histograms of acriflavine Feulgen-stained CHO cells in exponential growth, synchronized in the early S phase by double thymidine block, arrested in G1 phase by isoleucine deprivation, or in mitosis by mitotic shake off. This figure was used at the introduction of every presentation by one of us (HAC) to provide validation of the techniques. Still, some skeptics in the audience often expressed doubts whether the cell cycle was indeed being assayed by FCM. However, convinced that the method was reliable and accurate, we used the acriflavine-Feulgen procedure for analysis of cells treated with a variety of chemotherapeutic agents supplied by the National Cancer Institute (NCI). The first paper demonstrating this approach appeared in 1972 (12). Independently, in Europe, Göhde and Dittrich (13) also used FCM to analyze cytostatic affects of antitumor drugs. Several improved DNA cell-staining techniques were developed in the early 1970s that led to methods for cell cycle analysis that remain in widespread use today. These methods used the phenanthridine fluorochromes EB and PI. Both dyes intercalate into ds DNA and RNA with a fluorescence enhancement 20–30 times more than the unbound dye. EB was used by Dittrich et al. (14) in combination with fluorescein isothiocyanate (FITC) to stain both DNA and protein, respectively, in cells following enzymatic removal of RNA during the staining reaction. Subsequently, this technique was modified by the substitution of PI for EB (15). This was a novel cytological use of PI and, since the dye was not yet commercially available, a sample was obtained gratis from Dr. B. Hudson and colleagues, who had synthesized PI for use in procedures for the isolation of closed circular DNA (16). Both EB and PI are cell-impermeant and ethanol fixation was required for the above procedures. However, Krishan (17) later used PI in a hypotonic citrate solution, which essentially disrupted the cell membrane so that fixation and RNase treatment of the cell samples was not required. Data obtained from analysis of cell populations obtained from solid tumors grown subcutaneously in mice and stained using this procedure is shown in Figure 2. The hypotonic method was simple and rapid; these were the virtues that contributed to its popularity. The protease/detergent method subsequently developed by Vindeløv et al. (18) provided consistently high resolution of DNA content measurement (lower CV) and was more often used for analysis of clinical samples. DNA content histograms obtained from B16 melanoma and Lewis lung carcinoma cell populations stained with PI in hypotonic citrate (17). Another rapid cell staining procedure resulted from somewhat incident findings of one of us (HAC), obtained while studying the effects of the fluorescent antibiotic mithramycin (MI) on cell cycle progression. Subsequent investigations demonstrated MI-specificity for DNA in single cells, and a procedure was developed using MI in a simple solution containing phosphate buffered saline (PBS), magnesium chloride, and 25% ethanol (19). Cells were thereby permeabilized and stained in one step, and cell cycle analysis could be performed in a total of 20 min. The MI procedure received substantial application in the 1970s, but the requirement for a 5.0-W argon laser to provide adequate 457-nm excitation of the fluorochrome has limited it usage today. The DNA content histogram in Figure 3, obtained using the MI staining technique, shows the Dean and Jett (20) computer-fit modeling used to obtain the percentage of cells on S phase from the histogram. For comparison, Figure 3 also shows the percentage of S phase cells obtained by 3H thymidine labeling and autoradiography. A combination of MI and EB as DNA stain, introduced by Barlogie et al. (21), was reported to provide better DNA content resolution than each of these dyes alone (22). Chromomycin A3, an antibiotic with a chemical structure similar to MI, was used by Jensen (23) for cell cycle analysis of human gynecological samples. DNA histograms of CHO cells stained with MI (19). The percentage of cells in S phase as obtained by the computer fit model of Dean and Jett (20) from FMF, was compared to the percentage of cells obtained by pulse-labeling with tritiated thymidine and autoradiography. The UV-excited fluorochrome DAPI was introduced to FCM by Stöhr et al. (24) and Göhde et al. (25). Among several DNA fluorochromes compared, binding of DAPI to DNA was shown to be the least affected by differences in composition and structure of nuclear chromatin (26). DAPI, therefore, appears to be the preferred fluorochrome for quantitative analysis of cellular DNA content in different cell types, regardless of structure (degree of condensation) of nuclear chromatin. In concordance with this observation (26), the highest resolution of DNA content histograms (the lowest CV values of the mean DNA content of uniform cell populations) was reported for the cells, including spermatozoa, stained with DAPI (CV = 0.5–1.0%) (27). Early studies utilized impermeant fluorochromes that required cell fixation—a step incompatible with cell viability. Hilwig (28) introduced Hoechst 33258 and Hoechst 33342 as supravital DNA fluorochromes and Arndt-Jovin and Jovin (29) used them in FCM and to sort live cells on the basis of cellular DNA content. The Hoechst dyes require excitation in the UV light range. Recently, Smith et. al. (30) have shown that another fluorochrome, DRAQ5, also is able to stain live cells. This far-red fluorescing dye can be excited in the 488–647 nm range. However, DRAQ5 appears to be fairly toxic, so the long-term viability of the stained cells is limited. Despite that limitation, DRAQ5 is expected to find a host of applications, since, unlike Hoechst dyes, it does not require UV excitation. The success of permeant DNA fluorochromes that can be used for live cells depends on either nonfluorescent binding to RNA and other polyanions, or not binding. Hoechst 33342 binds to RNA, but does not fluoresce (Jacobberger, unpublished). Knowing how difficult it is to isolate intact cells or cell nuclei from fresh tissue, one has to be an extreme optimist to attempt to isolate cells or nuclei from paraffin-embedded tissues. Certainly such an optimist was Hedley et al. (31), who developed the methodology that allows one to subject cell nuclei isolated from paraffin blocks to FCM analysis. This technique opened unlimited possibilities to analyze archival specimens stored in pathology departments for retrospective studies. There are some limitations to this methodology, primarily related to variability in fixation procedure (e.g., duration, temperature, formaldehyde strength, specimen size, and tissue type), which in turn affect DNA stainability after nuclei isolation. Nevertheless, the methodology became widely used and helped to attract legions of pathologists, who used FCM primarily to explore whether the frequency of S phase cells (indicative of a higher fraction of cycling cells) or the presence of more than one stem line in cancers (DNA-aneuploidy) may be prognostic indicators. Prior to the development of DNA histogram deconvolution algorithms to analyze the FCM data, the investigators had to improvise and develop their own approaches. The most common was integration of the cell count by computer upon setting the gates for G1 versus S versus G2/M cell populations "by eye." Accuracy, objectivity, and reproducibility of such an approach were debatable, and reviewers of these papers had an open field to criticize the analysis. Needless to say, often the coauthors had different opinions and vehemently argued between themselves as to where to set the gates, particularly when cell populations were synchronized and the histogram did not show the distinct G1 and G2/M peaks. Another approach was to copy outlines of the histogram from the image on an oscilloscope onto uniform thickness filter paper. The peaks and ridges of the histogram related to G1 versus S versus G2/M cells were then cut off and weighted. Their weight represented the integrated number of cells in respective phases of the cell cycle. Obviously, sharp scissors and accuracy of the balance were as essential for precision of DNA measurement as good histochemistry and a good flow cytometer. One of us (JWJ, Ph.D. thesis) used this paper cutting technique to measure the cell cycle stages of malaria parasites. This work was rejected from a parasitology journal, with a reviewer stating that the paper cutting method was "unsophisticated." When the ModFit (Verity Software House, Topsham, ME) flexible modeling software was introduced, the same data were analyzed in a "sophisticated" manner and was subsequently published (32). The results obtained by the two methods were highly correlated and provided identical biological information. Reproduction of the DNA histogram image that appeared on the oscilloscope screen of the pulse-height analyzers connected to the early FCM system was time consuming and expensive. A hooded Polaroid camera was used to take a photograph of the histogram and a protractor was then used to expand and scale the histograms onto graph paper. This figure was then provided to a draftsman who drew and labeled the axes and reproduced the histogram with printer's ink. Photographs of the figure provided the 8 × 10 inch glossy required for the publication, and the 35-mm slide for the presentation. The process sometimes took one to two weeks. The progress in computer capability and mathematical proficiency of some of our colleagues who were interested in the cell cycle led to the development of algorithms to deconvolute DNA histograms. For mammalian cells, Gaussian distributions were used to model the G0/1 and G2+M fluorescence distributions and either a polynomial (20) or a trapezoid (33) function was used to fit the S phase. A third, less popular fitting routine that works well for synchronized cells, uses multiple Gaussian distributions to fit the S phase In all used the S phase are with Gaussian This the statistical accuracy of S phase fraction computer in use are ModFit by (Verity Software House, Topsham, ME) (33) and by of These have to estimate the of distributions that can be to and nuclei that have cut These provide accurate measurements from data that can be by which are often e.g., in samples of nuclei from paraffin-embedded blocks or For a time, DNA analysis was on clinical with many results that the presence of stem and high S phase were of for many However, the simple technique of and staining cells was difficult for many laboratories to and many studies were reported that were The of this was a large in the clinical value and of single DNA analysis. This resulted in an of that was to a of interested seeking to either the or at least find the was that DNA content measurements prognostic information for several a later that DNA content measurements for and cancers not be that many studies were in that multiple S phase were used for high and S phase and were and that there was a of studies et al. showed both of S phase and as well as the improved prognostic of DNA content measurements on The approach was analysis with 10 to procedures that were developed on one large retrospective = then applied to two and with DNA content analysis made it to G0/1 versus S versus G2/M cells, it could not between cells the same DNA content such as cells versus G1 or versus M cells. of these cells is The first cytometric methods that were able to identify these cells utilized the fluorochrome that under the of staining can stain versus ds binds to ds by and this binding in fluorescence with on the other of the which then after of RNA, can stain DNA versus RNA over of total cellular RNA is and cells have compared to their cycling G1 following staining of DNA versus RNA with cells were as the cells with RNA content (Fig. 4). It was also to early G1 cells from the G1 cells, based on a in RNA content that the cells S phase in some cell the cells that were had an S or a G2/M DNA such cells also had RNA content and could be from cycling S and G2/M cells The same cell cycle were subsequently using Hoechst 33342 and as DNA and RNA of and G2/M of the cell cycle of based on staining of DNA and RNA with The RNA content of cycling G1 cells compared to cells is It is also that G1 cells enter S phase only when the of RNA has this from cells for The first cytometric method that one to from M cells also utilized However, in this technique, RNA was by prior of cells with RNase and DNA was by or When such cells were stained with the ds DNA the fluorochrome by fluorescing while the had fluorescence It had that DNA in chromatin of M cells is more to when or compared to DNA of or cells. DNA in chromatin of cells was more to compared to DNA of G1 cells. This method, one to all five cell cycle and M (Fig. DNA of and form a uniform Early during cells to proliferation are by and fluorescence of and M cells can be on the of The shown in was treated with for 8 to cells in the in frequency of M cells and the in cells. The in in DNA to in chromatin the fluorochrome that was able to probe two cell such as cellular RNA content and of DNA each with measurement of DNA has become a used in studies of the cell cycle. The cell cycle by this dye in number of or of chromatin as the cells the cycle from and through and As has of using flow cytometry to DNA replication can only be by the investigators who used 3H-thymidine autoradiography for the same progress in this The first approach was based on the that fluorescence of Hoechst to DNA is in cells that incorporated the thymidine The cells, therefore, could be based on the in Hoechst compared with their DNA content. DNA staining with Hoechst with the dye fluorescence is by the incorporated such as made it to identify cells with analysis of the cell cycle of the measured cell were using and to estimate many of the cell cycle kinetics One of the distinct of this method for studies was that multiple could be The of this method was improved by using MI instead of and by using the of of Hoechst from as a (Fig. DNA content and and the MI frequency histograms and the for and for CHO cells treated with for The of in S phase cells can be in and the significant MI differences in S phase cells is in for Because Hoechst fluorescence of to DNA is also by by with of M cells based on DNA it was to the from M cells This approach opened a possibility of the use of FCM for the fraction of labeled mitosis to analyze cell cycle kinetics (3, 4). The approach to DNA replication by flow cytometry was based on of In this method, following DNA by M or the incorporated to The is most or labeled with When the DNA are counterstained with a fluorescing fluorochrome, such as the distributions both cellular DNA content and the presence of incorporated of the incorporated the step of DNA This treatment often cell and by many of other The approach developed for the DNA step and is with a use of other This is the by method which on of the cells with UV light to at DNA that the incorporated DNA by are then labeled with the by the as in the to DNA in cells (Fig. are commercially e.g., that on this methodology. of by the method The cells were with for their DNA was by to UV light DNA to UV and the DNA were labeled with using DNA was counterstained with PI. shows cells that were not with Because of a high to ratio by and because a than UV is the of has become the predominant methodology to assess DNA and in to kinetics of cell cycle progression. Several mathematical models have developed to the cohort of cells with through different cell cycle phases to obtain the value in the is the method developed by et al. to measure duration of S phase and time of cells, from a single sample at a in time after in vivo of As cell such as RNA content and of chromatin were to be to from cycling cells and identify several of the cell cycle. The in probe development were focused on expected to higher of the cell cycle progression prior to of and to have and on different cell primarily to assess their as and prognostic of cell of these were isolated