Flow cytometric localization within the cell cycle and isolation of viable cells following exposure to cytotoxic agents.

Viable cell sorting, based on flow cytometric analysis of DNA content and cell volume, was used to evaluate the cycle position and survival potential of Adriamycin (AdR)-treated or 1-beta-D-arabinofuranosylcytosine (ara-C)-treated CHO cells. Drug-treated cells initially stained with the vital, DNA-specific fluorochrome, Hoechst 33342, were analyzed for DNA content and volume, and sorting "windows" were established for subsequent sorting of duplicate unstained cell samples based only on cell volume. Another portion of the cell sample was fixed in ethanol, and stained with three fluorochromes for correlated flow cytometric analysis of DNA, RNA, and protein. Similarities in the viable cell volume distributions and the protein content distributions of the ethanol-fixed samples provided a means for indirectly determining the DNA and RNA contents of the sorted cells. Three regions (S, L, and I) were selected in the cell volume distributions corresponding to the range of near normal cell size (S), larger than normal cell size (I), and the extremely large cells (L). Adriamycin-treated or ara-C-treated cells sorted from the S region had survival values, respectively, 46 times and 7 times greater than the abnormally large cells in region L. Cells from the S region also respectively survived 14-fold (AdR-treated) and 7-fold (ara-C-treated) greater than the cells sorted from the I regions. RNA content levels for cells within the L region were three times and two times greater, respectively, than the AdR-treated and Ara-C-treated subpopulations in the S regions. Survival of subpopulations of G2-arrested, AdR-treated cells (I and L regions) was better correlated with relative abnormality in cell size than with position in the cell cycle. In addition to providing further support for the validity of the "balanced growth hypothesis," the results of this study suggest that two-parameter DNA content and cell volume measurements would be extremely useful for providing general guidelines for judging the effectiveness of therapy, especially in clinical diagnoses where cell sorting is impractical or impossible. From these analyses the frequency and cycle position of cells resistant to therapy can be estimated. Such information would be particularly useful for rapidly detecting drug-resistant cells and design of subsequent therapeutic regimens.

Differential fluorochromasia of human lymphocytes as measured by flow cytometry.

Peripheral human lymphocytes reacted with fluorescein diacetate and analyzed by flow cytometry produced a bimodal fluorescence distribution that was shown to be attributable to the differential staining of T and B lymphocytes. Lymphocytes were fractionated into rosetting (T cell) and nonrosetting (B cell) populations. Both subfractions were reacted with fluorescein diacetate and analyzed by flow cytometry. The rosetting fraction was more fluorescent than the nonrosetting fraction, and the analysis of an appropriate mixture of the subfractionated populations produced a fluorescence distribution very similar to that obtained with unfractionated lymphocytes.

Light scattering with stream-in-air flow systems.

Both forward angle and 90 degrees light-scattering measurements have been used for cell sizing with stream-in-air flow systems with very little theoretical base for the measurements. Mie theory calculations are compared with measurements on plastic microspheres. Detector response for homogeneous spheres is shown to be sensitive to refractive index.

Flow cytometric determination of radiation-induced chromosome damage and its correlation with cell survival.

Chinese hamster M3-1 cells were irradiated with several doses of X rays or alpha particles from 238Pu. Propidium iodide-stained chromosome suspensions were prepared at different times after irradiation; cells were also assayed for survival. The DNA histograms of these chromosomes showed increased background counts with increased doses of radiation. This increase in background was cell-cycle dependent and was correlated with cell survival. The correlation between radiation-induced chromosome damage and cell survival was the same for X rays and alpha particles. Data are presented which indicate that flow cytometric analysis of chromosomes of irradiated cell populations can be a useful adjunct to classical cytogenic analysis of irradiation-induced chromosomal damage by virtue of its ability to express and measure chromosomal damage not seen by classical cytogenic methods.

Coulter volume cell sorting to improve the precision of radiation survival assays.

A new method for measuring cell survival at low doses of ionizing radiation has been developed through the use of flow cytometric cell sorting on the basis of Coulter volume signals. The cell sorter is capable of deflecting a precisely known number of cells directly into culture dishes, thus eliminating any errors associated with cell dilution and volume sampling. The use of Coulter volume signals as the sorting parameter is shown to be noncytotoxic for a variety of cell lines. Comparison of radiation survival curves measured above the 10% survival level by either the cell sorter or standard dilution assay demonstrates the increased precision of the cell sorter technique . Because of these advantages of cell sorting over conventional methods of plating cells, this technique has many applications in the field of radiation biology and other studies of cell survival.

Radiobiology of ultrasoft X rays. II. Cultured C3H mouse cells (10T1/2).

In the first paper of this series (Radiat. Res. 110, 396-412 (1987], using V79 cells, we reported that the relative biological effectiveness (RBE) of ultrasoft X rays was found to increase with decreasing energy, and the oxygen enhancement ratio (OER) was found to decrease with decreasing energy. In this report, we present RBE and OER results for 10T1/2 cells that are known to grow uniformly flat and are considerably thinner than V79 cells. Thus the variation in dose across the cell nucleus is considerably reduced. The OER results agree well with our earlier V79 results. However, the RBE values for 10T1/2 cells compared to V79 cells are systematically less for all soft X rays and especially for 0.28 keV carbon-K (1.3 compared to 3.4 for V79 cells). Some plausible explanations are presented to reconcile the apparent discrepancy between V79 and 10T1/2 results.

Radiobiology of ultrasoft X rays. III. Normal human fibroblasts and the significance of terminal track structure in cell inactivation.

Ultrasoft characteristic X rays from carbon (0.28 keV) are severely attenuated as they pass through biological material, causing a nonuniform distribution of dose to cell nuclei. Complications of studying ultrasoft X rays can be minimized in this context by using cells with very thin cytoplasm and nuclei (e.g., less than the attenuation length of the X rays), and which exhibit a more nearly exponential dose response to cell killing, such as normal human fibroblasts compared with V79 cells. Using this cell system, we report the relative biological effectiveness (RBE) of A1-K and C-K X rays to be near unity. Previous studies of cell inactivation by characteristic carbon X rays gave RBEs of 3 to 4, supporting the idea that localized energy depositions from secondary electrons and primary track ends represent the principal mode of biological action for other low-LET radiations. In part, the reported high RBEs result from the use of mean dose to describe energy deposited within the cell nuclei by these poorly penetrating radiations. Implicit in the use of mean dose is that cellular damage varies linearly with dose within a critical target(s), an assumption that is of questionable validity for cells that exhibit pronounced curvilinear dose responses. The simplest interpretation of the present findings is that most energy depositions caused by track-end effects are not necessarily more damaging than the sparsely ionizing component.

Radiobiology of ultrasoft X rays. IV. Flat and round-shaped hamster cells (CHO-10B, HS-23).

The results reported earlier in this series indicated that the relative biological effectiveness (RBE) of ultrasoft X rays decreases with decreasing cell thickness, approaching unity for the thinnest cells used, plateau-phase human skin fibroblasts (HSF). The possible dependence of RBE on the configuration of the cell nucleus is investigated further in this paper using two CHO cell lines that attach well and have similar intrinsic radiosensitivities to 60Co gamma rays. One of the lines forms monolayers similar to V79 cells, while the other remains more spherical during growth. We find an increasing RBE with decreasing X-ray energy for both of these cell lines, consistent with our results using V79 cells. Also consistent with our results obtained with 10T1/2 and HSF cells, we find an increasing RBE with increasing cell thickness. The possible dependence of RBE on radiosensitivity and the use of the concept of mean dose for ultrasoft X rays is discussed.

Mutational extinction induced by sequential X irradiation and actinomycin D treatments in Chinese hamster ovary cells.

The interactions of sequential X irradiation and actinomycin D (AMD) treatments for mutagenesis to 6-thioguanine resistance were investigated in CHO cells. Cells were exposed to single doses of X rays followed immediately by 1-h treatments with 0.1 or 1 microgram/ml AMD. X Rays alone induced mutagenesis which increased monotonically with dose to at least 8 Gy. AMD-treated control cultures showed slight to moderate cytotoxicity and little induced mutation. X Rays followed by AMD treatment produced bell-shaped mutagenesis dose-response curves with maximal mutation at approximately 5 or 4 Gy for 0.1 or 1.0 microgram/ml AMD, respectively. Induced mutation frequencies then fell to a negligible level at fractional survival levels below 0.10 for either combination treatment. Application of a stochastic Poisson distribution model to these data led to the prediction that two possible components govern induced mutation frequencies. First, X ray +AMD induced mutations may be depleted progressively with dose from the surviving populations by selective lethality, which we term mutational extinction. Second, X ray +AMD treatments were calculated to induce potentially much greater than additive mutagenesis. However, due to the overriding mutational extinction effect, most of these mutations are not recovered as viable colonies. These studies suggest that AMD binding to DNA immediately following irradiation may cause considerably enhanced mutagenic and often lethal DNA damage, and that mutational extinction may occur because these types of damage are statistically correlated in a sensitive subpopulation of exponentially growing CHO cells.

Radiobiology of ultrasoft X rays. I. Cultured hamster cells (V79).

Ultrasoft X rays (approximately less than keV) provide a useful probe for the study of the physical parameters associated with the induction of biological lesions because the spatial scale of their energy depositions is of nanometer dimensions, comparable to that of critical structures within the cell. We report on cell-killing experiments using cultured hamster cells (V79) exposed to carbon K (0.28 keV), aluminum K (1.5 keV), copper K (8.0 keV), and 250 kVp X rays, under oxic and hypoxic conditions, and as a function of cell-cycle phase. Our principal results are: RBE increases with decreasing X-ray energy; OER decreases with decreasing X-ray energy; and cell-cycle response is similar for all X-ray energies. Our RBE results confirm earlier observations using ultrasoft X rays on mammalian cells. The shapes of fitted curves through the data for each energy are statistically indistinguishable from one another, implying that the enhanced effectiveness is purely dose modifying. The results reported herein generally support the view that single-track effects of radiation are predominantly due to very local energy depositions on the nanometer scale, which are principally responsible for observed radiobiological effects.

Viable sorting of intact multicellular spheroids by flow cytometry.

A flow cytometric method has been developed for sorting viable, intact multicellular spheroids in order to obtain uniformly-sized populations with diameters in the range of 50-100 microns. A FACS II instrument was modified for this purpose by installing a 200-microns-diameter exit orifice and by making adjustments in the sheath flow, oscillator frequency, and number of droplets sorted. Polystyrene microspheres (44 and 88 microns diameter) and 41-96-microns-diameter spheroids could be sorted and recovered with 70-100% efficiency, an improvement over previous reports. Unstained, viable spheroids were simultaneously analyzed for small-angle forward light scatter, 90 degree light scatter, and autofluorescence using a 488-nm laser operating at 100 mW. Analysis of the data demonstrated a considerable variation in both the 90 degrees light scatter and the autofluorescence signals for a given forward angle light scattering signal. By setting narrow sort windows on the forward angle light scattering signal and either the 90 degree light scatter or autofluorescence signals, uniformly spherical spheroid populations could be recovered. These sorted populations had coefficients of variation of the mean diameter in the range of 5-9%. This represents a variation of less than one cell diameter, and is a major improvement over any other technique. There was no significant difference in the subsequent growth rates of sorted spheroids compared to the unsorted spheroids. This technique will apply when uniform populations of small spheroids are required, such as investigations of the contact effect or in the initiation of growth curve studies.

Rapid assay for cell age response to radiation by electronic volume flow cell sorting.

A new technique is described for measuring cell survival as a function of cell cycle position using flow cytometric cell sorting on the basis of electronic volume signals. The sorting of cells into different cell age compartments is demonstrated for three different cell lines commonly used in radiobiological research. Using flow cytometric DNA content analysis and [3H]thymidine autoradiography of the sorted cell populations, we demonstrate that the resolution of the age compartment separation is as good as or better than that reported for other cell synchronizing techniques. The variation in cell survival as a function of position in the cell cycle after a single dose of radiation as measured by volume cell sorting is similar to that determined by other cell synchrony techniques. This new method has several advantages, including: no treatment of the cells is required, thus, this method is noncytotoxic; no cell cycle progression is needed to obtain different cell age compartments; the cell population can be held in complete growth medium at any desired temperature during sorting; and a complete radiation age-response assay can be plated in 2 h. The application of this method to problems in radiobiology and chemotherapy is discussed, along with some of the technical limitations.

Flow cytoenzymology of the early differentiation of mouse embryonal carcinoma cells.

Dual parameter flow cytoenzymology was used to detect biochemical differentiation of embryonal carcinoma cells, the undifferentiated, multipotent stem cells of teratocarcinomas. With the use of fluorogenic substrates, two enzyme systems, alkaline phosphatase (EC 3.1.3.1.) and carboxyl esterase (EC 3.1.1.), were studied. Embryonal carcinoma cells passaged in vitro for several years retained high alkaline phosphatase activities similar to those of embryonal carcinoma cells in embryoid bodies grown in vivo. Similar to the embryonal carcinoma cells in vivo, the in vitro embryonal carcinoma cells were capable of giving rise to progeny with greatly decreased levels of alkaline phosphatase. The embryonal carcinoma cell alkaline phosphatase was inhibited by 1-p-bromotetramisole, suggesting a relationship between this enzyme and somatic, nonintestinal alkaline phosphatase isoenzymes. Determinations of esterase activities in viable teratocarcinoma cells showed that prior to any evidence of morphologic differentiation, the embryonal carcinoma cells are quite heterogeneous with regard to esterase activities.

A simple electronic volume cell sorter for clonogenicity assays.

A single-parameter electronic volume flow cell sorter that can be easily and inexpensively constructed using existing technology is described. The instrument is designed for ease and flexibility of operation, including such features as a large open area for recovering sorted cells into a variety of dishes or vessels; a remote, electrically activated fluidics system; a mechanism for heating or cooling samples during sorting; a simple arrangement for monitoring and adjusting the sorting control parameters; and an interface to a standard IBM personal computer for data acquisition, analysis, and control of the sorting windows. Several researchers in our laboratory now routinely use this sorter for plating precise numbers of cells directly into culture dishes in an aseptic manner for clonogenicity assays. The instrument can sort cells at rates of up to approximately 2,000 per second with greater than 80% sorting efficiency and no cytotoxicity. An advantage of this system is that the sorting windows can be set to exclude acellular debris and include either the entire cell volume distribution or a subset thereof. Applications of the instrument are detailed, including 1) precise cell plating for low-dose survival studies, 2) separation of cells into age compartments, and 3) rapid inoculation of single cells into multiwell dishes for cloning studies. Advantages of this technology for cell survival studies are detailed, along with some limitations to its applicability.

A method for staining 3T3 cell nuclei with propidium iodide in hypotonic solution.

A modification of the propidium-iodide hypotonic sodium citrate method has been developed specifically for high-resolution staining of mouse 3T3 cell nuclei for analysis by flow cytometry. The method employs a brief treatment of cells at 37 degrees C with Triton X-100 and RNAse in the presence of propidium iodide in hypotonic sodium citrate, followed by restoration to isotonicity with NaCl. The average CV obtained for the G1 peak was 3.5%, and the samples were stable for 1-2 weeks at 4 degrees C. Compared to this technique, previously described propidium iodide-staining methods gave poor resolution with 3T3 cells.

Sensitivity to X-irradiation in relation to cell size of CHO cells synchronized in early G1.

A population of line CHO Chinese hamster cells was synchronized by mitotic selection and allowed to enter early G1, after which the largest and smallest cells in the population were sorted, irradiated, and their viability determined. Despite sizeable differences in volume, metabolic capability and cell cycle progression rates, an equivalent level of survival was obtained for the two populations, indicating that the factors responsible for the volume, metabolic and progression heterogeneity do not contribute greatly to radiation sensitivity.

Combined flow and absorption DNA measurements of [3H]TdR-labelled tumour cells. I. Studies of MCa-11 cells grown as tumours in vivo and as exponential cultures in vitro.

Flow cytometry of cellular DNA content provides rapid estimates of DNA distributions, i.e. the proportions of cells in the different phases of the cell cycle. Measurements of DNA alone, however, yield no kinetic information and can make it difficult to resolve the cell cycle distributions of normal and transformed cells present in tumour biopsy specimens. The use of absorption cytophotometry of the Feulgen DNA content and [3H]TdR labelling of the same nuclei provides objective criteria to distinguish the ranges of DNA content for G0/G1, S, and G2/M cells. We now report on a study in which we combined flow and absorption cytometry to resolve the cell cycle distributions of host and tumour cells present in biopsy specimens of MCa-11 mouse mammary tumours labelled in vivo for 0.5 hr with [3H]TdR. A similar analysis of exponential monolayer cultures, labelled for 5 min with [3H]TdR under pulse-chase conditions, revealed a highly synchronous traversal of almost all cells through the different phases of the cell cycle. Combination of the flow and absorption methods also allowed us to detect G2 tumour cells in vivo and a minor tumour stem-line in vitro, to show that these two techniques are complementary and yield new information when they are combined.

Use of DiO-C5-3 to improve Hoechst 33342 uptake, resolution of DNA content, and survival of CHO cells.

Chinese hamster cells (line CHO) stained with either 9 microM Hoechst 33342 (HO) alone or in combination with the membrane potential fluorochrome DiO-C5-3 (DiO) were analyzed using uv laser powers between 25 and 500 mW and sorted for determination of survival by a colony formation assay. The combination of HO-DiO increased fluorescence twofold and provided coefficients of variation (CVs) as low as 3.0% under conditions where viability of cells, even at 500 mW excitation, was unaffected. HO-stained cells yielded CVs of about 8.5% and survivals of approximately 90% under similar analytical conditions. At laser powers of 25 mW, CV values for HO-DiO-stained populations were 4.0% compared to 9.4% for HO-stained cells. Results with another membrane potential dye, rhodamine 123 (R 123), in combination with HO showed no improvement compared to HO-stained cells. No preferential, cell cycle phase-specific killing was observed in either the HO- or HO-DiO-stained populations. CVs of human skin diploid fibroblasts stained with HO or with HO-DiO were comparable over the entire laser power range; however, percentage survival was slightly higher for the HO-DiO-stained populations when analyzed and sorted at the higher power (400-500 mW) range. Long-term cultures of sorted CHO-K1 subpopulations, differing in DNA ploidy, were established from HO-DiO-stained cells. Advantages of this new staining procedure include improved DNA content resolution (low CV values) and the potential use of less expensive FCM uv laser systems coupled with less perturbing excitation powers.

Reversible accumulation of plant suspension cell cultures in g(1) phase and subsequent synchronous traverse of the cell cycle.

The induction of DNA synthesis in Datura innoxia Mill. cell cultures was determined by flow cytometry. A large fraction of the total population of cells traversed the cell cycle in synchrony when exposed to fresh medium. One hour after transfer to fresh medium, 37% of the cells were found in the process of DNA synthesis. After 24 hours of culture, 66% of the cells had accumulated in G(2) phase, and underwent cell division simultaneously. Only 10% of the cells remained in G(0) or G(1). Transfer of cells into a medium, 80% (v/v) of which was conditioned by a sister culture for 2 days, was adequate to inhibit this simultaneous traverse of the cell cycle. A large proportion of dividing cells could be arrested at the G(0) + G(1)/S boundary by exposure to 10 millimolar hydroxyurea (HU) for 12 to 24 hours. Inhibition of DNA synthesis by HU was reversible, and when resuspended into fresh culture medium synchronized cells resumed the cell cycle. Consequently, a large fraction of the cell population could be obtained in the G(2) phase. However, reversal of G(1) arrested cells was not complete and a fraction of cells did not initiate DNA synthesis. Seventy-four percent of the cells simultaneously reached 4C DNA content whereas the frequency of cells which remained in G(0) + G(1) phase was approximately 17%. Incorporation of radioactive precursors into DNA and proteins identified a population of nondividing cells which represents the fraction of cells in G(0). The frequency of cells entering G(0) was 11% at each generation. Our results indicate that almost 100% of the population of dividing cells synchronously traversed the cell cycle following suspension in fresh medium.

Oncogenes and linkage groups: conservation during mammalian chromosome evolution.

Proto-oncogenes, which represent the cellular progenitors of the transforming genes harbored by acute transforming oncogenic retroviruses, have been highly conserved during vertebrate evolution. In this report, we have assigned experimentally a subset of proto-oncogenes (SRC, ABL, FES, and FMS-all related to the SRC family) to Chinese hamster chromosomes by Southern filter hybridization analyses of DNAs isolated from both somatic cell hybrids and flow-sorted hamster chromosomes. These results demonstrate that several autosomal linkage groups containing proto-oncogenes originated prior to the radiation and speciation of mammals and have remained remarkably stable for nearly 80 million years.