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Two flow cytometric assays for cellular RNA, two-step acridine orange (TSAO) and propidium iodide (PI), were compared with each other and with ultraviolet (uv) spectrophotometry of RNA to determine their ability to quantitate cellular RNA and to differentiate between proliferating and quiescent (Q) cells. The model system used for these comparisons was cells from unfed cultures of two mouse mammary tumor lines designated 66 and 67. The growth kinetics of unfed 67 and 66 cells were characterized by a decrease in cellular RNa (a factor of 2) with the decrease occurring throughout the entire population of cells as they entered plateau phase. The time course of the RNA decrease as monitored by the PI assay closely corresponded to that observed by uv spectrophotometric measurements. The TSAO assay, however, agreed with the uv spectroscopy and PI assays on the extent of the RNA decrease but showed no decrease in RNA until 24 hours after the other assays indicated that a significant decrease had occurred. The plateau cells from 67 and 66 unfed cultures had a greatly lowered RNA content and were predominately (greater than 97%) in the G1 phase of the cell cycle in terms of DNA content. However, when compared with exponentially growing cells, they were a distinct Q population. This distinction could be observed using either the TSAO or the PI assay. The TSAO assay provided better resolution of the Q population and has the added advantage of giving DNA distribution simultaneously. Therefore, both flow cytometric assays are particularly useful in this system; the TSAO for monitoring the Q population and the PI for determination of kinetic changes in total cellular RNA.

The effects of actinomycin-D and 3'-deoxyadenosine (cordycepin) on the steroid-mediated induction of tyrosine aminotransferase (TAT) synthesis have been reexamined in view of recent reports that the primary inhibitory action of these compounds may affect synthesis of proteins as well as RNA. The present results confirm that cordycepin blocks the steroid-mediated induction of TAT in rat hepatoma cells (HTC), but unlike actinomycin-D, cordycepin neither increases nor maintains the levels of TAT found in HTC cells preinduced with dexamethasone. Indeed, cordycepin added to preinduced cells, either in the presence or absence of steroid, causes a prompt decline in TAT activity. These data also confirm that both actinomycin-D and cordycepin have an early inhibitory effect on protein synthesis, but the cordycepin effect is observed sooner and the extent of inhibition is greater. When actinomycin-D and cordycepin are added simultaneously to preinduced cells with the steroid removed, the actinomycin-td produced maintenance of preinduced levels of TAT persists. Also, the inhibition of protein synthesis in cultures with both inhibitors approaches that for the cells treated with actinomycin-D alone instead of cordycepin alone. These data suggest that cordycepin inhibits TAT synthesis in preinduced cells by its inhibition of protein synthesis, and this inhibitory effect of cordycepin is blocked by actinomycin-D. It is possible that actinomycin-D does this by preventing the incorporation of cordycepin into RNA. However, regardless of the correctness of this speculation, the multiple effects of cordycepin indicate that this inhibitor cannot be used either to prove or rule out the post-transcriptional model for regulation of gene expression. Also, this requirement that protein synthesis must continue in order to maintain pre-induced levels of TAT is inconsistent with the assumption that the maintenance of these induced TAT levels by actinomycin-D is due to inhibition of TAT degradation.

The fluorescence properties of Hoechst 33342 (HO 33342) were examined with plasmid pBR322 in the supercoiled (Form I) or relaxed covalently closed circular (Form Io) conformation in order to determine whether qualitative or quantitative differences in fluorescence properties might provide an assay for topological states of DNA. It was found that HO 33342 exhibited a 30% greater fluorescence intensity with Form I pBR322, independent of the dye or DNA concentration. As the dye to DNA ratio was increased, a red shift of approximately 8 nm was observed for HO 33342 complexed with Form I or Form Io. The red shift in fluorescence emission occurred at higher HO 33342 concentrations with Form I vs. Form Io DNA; however, when Form I and Form Io were mixed in various proportions, neither the fluorescent intensity differences nor the HO 33342 concentration at which the wavelength shift occurred could be used to quantitate the relative proportions of topological states present. These results suggest that although the fluorescence properties of HO 33342 complexed with Form I DNA are different than those of HO 33342 complexed with Form Io DNA, the fluorescence assay is not sufficiently sensitive to quantitatively discriminate among a mixture of DNA in various topological states.

The endogenous thiols (PSH, protein sulfhydryls; NPSH, nonprotein sulfhydryls; and GSH, glutathione) were measured in the 66 and 67 murine carcinoma cells growing under different physiological conditions in vitro (e.g., proliferation, P; nutrient-deprived quiescence QI; and QI cells stimulated by refeeding the monolayer in situ and assayed 4 (St4) and 14 (St14) h later). The aerobic radiation response was also studied as a function of the physiological state and thiol concentration. The changes in PSH levels suggest that the proportion of thiol-containing proteins changed whenever the cells were in transition between different physiological states (e.g., when QI cells were stimulated by refeeding, the proportion of PSH was elevated dramatically over either QI or P cells). The NPSH and GSH levels were both down significantly in the QI vs. P cells as was the total thiol level (PSH plus NPSH). Fourteen h but not 4 h after stimulation, the NPSH and GSH levels had returned to or exceeded the P-cell levels. Also, the proportion of GSH in the NPSH fraction varied as a function of the physiological state. The 66 and 67 QI cells were both more radiosensitive than the respective P cells. Also, the 66 cell radiation-induced cytotoxicity had returned to the P response by about 4 h after refeeding but the stimulated 67 cells had not. However, no overall correlation was apparent between the various aerobic radiation responses and the pool sizes of either the total thiols or of the various subsets of thiols. The depressed total thiol level and the increased radiosensitivity of the QI cells could represent a cause-and-effect relationship or these parameters could be independent phenomena only related indirectly through the reduced metabolic activity of the quiescent cells.

The problems encountered in studying the heterogeneity of cells in solid tumors is reviewed with emphasis on the role of various analytical cytometric assays for studying both the biology and the dynamics and proliferating, quiescent and dead malignant cells in vitro and in vivo. Due to advances in cytometric technology, many interesting in vitro studies on tumor cells heterogeneity have been and will be conducted over the next several years. For example, the acidic acridine orange staining of HeLa cells in suspension culture does readily discriminate between proliferating and quiescent cells. Some of these assays have been and others will be extended to in vivo studies. However, it is obvious that either the current analytical cytometric techniques must be modified and refined to permit better resolution for the complex situation in vivo or other new analytical cytometric techniques will have to be developed before many interesting studies on tumor cell heterogeneity in vivo can be addressed with reasonable efficiency.

The DNase I digestion kinetics of DNA in isolated nuclei (from HeLa or murine mammary carcinoma, 67 cells) were assayed flow cytometrically by measuring the changes in ethidium bromide (EtBr) fluorescence following various digestion time intervals. The DNase I digestion curve was characterized by an initial 25-30% increase in fluorescence upon addition of the enzyme, a rapid reduction in fluorescence to approximately 50-55% in 30 minutes, and a limit digest of 45-50% beyond 45 minutes. Throughout digestion, the DNA histogram retained its characteristic bimodal shape, showing that histogram rearrangement was not responsible for the changes in EtBr fluorescence. Irradiation with 5 X 10(6) rads (137Cs-gamma-rays) or exposure to 50 mM EDTA caused an increase in EtBr fluorescence similar to that caused by DNase I, suggesting that DNA nicking and/or chromatin loosening were responsible for this increase. Residual DNA assayed by the solubilization of 14C-TdR (thymidine)-labeled DNA indicated a similar kinetic pattern without the initial increase. However, at the limit digest, the fraction of DNA remaining trichloroacetic acid (TCA) insoluble (10%) was smaller than that measured by loss of EtBr fluorescence (50% of initial, 40% of maximum). Part of this difference was due to the presence of TCA soluble DNA trapped within the nuclear matrix (15-20%). This trapped DNA was released when the digested nuclei were exposed to 0.5-1.0 M NaCl just prior to EtBr staining. Exposure of HeLa cells to three agents that are believed to cause changes in chromatin structure resulted in alterations in the DNase I digestion kinetics measured flow cytometrically.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth control is investigated in detail in fed and unfed HeLa-S3 suspension cultures. Two-step acridine orange staining and flow cytometric analysis indicated declines in cellular red fluorescence (proportional to RNA content) of 40-50% between exponential and plateau phase in both culture types. Cellular green fluorescence (DNA content) assessed simultaneously indicates an increment of cells with Gi-DNA content in plateau phase in the unfed cultures, while fed cultures show a brief increment in G1-phase cells in the transition phase followed by a recovery in plateau phase to a value similar to that of exponential cultures. Temporal declines in the 3H-thymidine pulse-labeling index are observed in both culture systems. These data along with the flow cytometry data indicate a distinct G1-arrest in the unfed plateau cultures and suggest a random arrest of cells about the cell cycle in fed plateau cultures. Acidic acridine orange staining and flow cytometric analysis furthermore indicate the occurrence of a quiescent population comprising approximately 345 of the total cells and consisting of both dead and viable cells in plateau phase unfed cultures. In contrast, fed plateau cultures show approximately 14% quiescent, mostly dead cells. Also, both culture systems show temporal declines in the clonogenic index and a longer cell-cycle transit time in plateau phase relative to exponential phase. These findings confirm earlier work which indicates that the environment has a profound influence on the mode of growth control for mammalian cells in vitro.