Enzymatic responses to radiation in cultured vascular endothelial and smooth muscle cells

Confluent monolayers of bovine aortic endothelial and smooth muscle cells were exposed to 0-5.0 Gy of 60Co gamma rays. From 0 to 72 hr after irradiation, the monolayer and culture medium were analyzed for cell (nuclei) number, DNA and protein content, the activities of angiotensin converting enzyme (ACE), lactate dehydrogenase (LDH), and superoxide dismutase (SOD), and LDH isoenzyme profile. Irradiated endothelial cells exhibited a time- and dose-dependent increase in cell detachment, decreased DNA and protein content and reduced ACE active per attached cell, increased LDH and SOD activities per microgram of DNA, and increased LDH activity in the culture medium. The latter was accompanied by a shift from LDH 1 to LDH 4 and 5. The release of LDH activity, observed after 0.5 Gy, was the most sensitive endothelial response, and occurred independent of or preceding cell detachment. Vascular smooth muscle cells contained two to three times more SOD activity than did endothelial cells and exhibited no significant responses to 5.0 Gy.     

In situ hybridization analysis of polyoma DNA replication in an inducible line of polyoma-transformed cells.

In situ hybridization has been used to study polyoma DNA replication in a clonal derivative of the inducible LPT line of polyoma-transformed cells designated as clone 1A. This study has shown that in clone 1A cultures maintained under normal growth conditions, 4–25 in 10,000 cells are spontaneously induced to synthesize polyoma DNA at an enhanced rate. In cultures exposed to mitomycin C (MMC), the percentage of induced cells remains approximately equal to the spontaneous level for 9 hr, and then increases for at least 24 hr up to 30–57% as more and more cells are asynchronously recruited to replicate the virus DNA.DNA reassociation kinetics and in situ hybridization have been used to determine the amount and distribution of polyoma DNA accumulated within clone 1A cells. These measurements have shown that a single induced cell in an MMCtreated culture produces 24,500 genome-equivalents of the virus DNA; second, that the average yield of virus DNA in a normally growing culture is only 41.7 genome-equivalents per cell; however, a single spontaneously induced cell in this culture produces as much virus DNA as an MMC-induced cell; third, that all the virus DNA molecules are found within the nuclei and many are clustered in aggregates containing up to 2000 genome-equivalents. We discuss the implications of these findings regarding the regulation of polyoma DNA replication in the LPT line.     

Quantitative oral exfoliative cytology. Effect of alcohol on normal buccal mucosa

OBJECTIVE: To assess the effect of chronic alcohol intake on the DNA distribution and cell area of normal oral mucosal cells. STUDY DESIGN: Smears were taken from clinically normal buccal mucosa of 50 patients attending an alcohol-problem service (i.e., chronic alcohol use) and average alcohol units per week recorded. DNA distribution histograms and total cell area values were then compared to those obtained from smears taken from a control group (which included social drinkers) of patients attending for routine dental treatment. Nuclear DNA content was assessed on 100 randomly selected, Feulgen-stained nuclei using a Seescan TV image analysis system, and total cell area was assessed on 50 Papanicolaou-stained cells using the Vids V image analysis system. RESULTS: The DNA distribution histograms were essentially diploid in appearance for the alcohol group, although there was an increase in nuclear DNA content in the occasional nucleus. A highly significant reduction in total cell area was found for the alcohol group when compared to the controls. CONCLUSION: The chronic ingestion of alcohol is associated with a reduction in total cell area but appears to have little effect on nuclear DNA content. Our previous research using the same technique showed that oral cancers are frequently nondiploid. Thus, a nondiploid DNA distribution histogram for smears taken from a clinically suspicious lesion in someone who consumes excessive amounts of alcohol is unlikely to be due to alcohol use alone and should indicate biopsy.     

Effect of Penicillin on the Multiplication of Meningopneumonitis Organisms (Chlamydia psittaci)

Although formation of infectious particles of meningopneumonitis organism in L cells was completely inhibited by 1 or more units of penicillin per ml, multiplication of reticulate bodies was observed, by light microscopy, in the presence of 200 units of penicillin per ml in stained smears of infected cells. When reticulate bodies were purified from cultures containing penicillin after 18, 30, and 45 hr of incubation, continuously increasing yields were obtained. When penicillin was added to infected cultures 0 to 15 hr after infection, no increase in infectivity was observed at 40 hr, but when antibiotic was added between 20 and 35 hr, partial synthesis of infectious particles was observed at 40 hr. On the other hand, removal of penicillin from an infected culture before 15 hr after infection did not affect the final yields of infectivity when assayed at 40 hr, but elimination of penicillin after 20 hr resulted in a decrease in infectivity. In suspensions of (32)P-labeled purified reticulate bodies grown in cultures containing penicillin and harvested 18 and 40 hr after infection, the (32)P distributions obtained by acid fractionation were similar to those of reticulate bodies from penicillin-free cultures. Cell membranes of reticulate bodies were also prepared from 40-hr cultures with penicillin. The size and shape of purified membranes, as seen by electron microscopy, and their amino acid compositions were similar to membranes prepared from reticulate bodies grown without penicillin, except that very small structures were observed in membranes from cultures containing penicillin. These results indicated that penicillin does not inhibit reproduction of reticulate bodies and formation of their cell membranes, but does inhibit the formation of elementary body cell envelopes.     

DNA ploidy abnormalities in rabbit preimplantation embryos are not increased by conditions associated with in vitro culture

Possible adverse effects of in vitro culture-associated physical factors were studied in 3- and 4-day-old rabbit embryos. Laboratory conditions were mimicked by exposure to visible light (320–740 nm, 1600 lx) or decreased temperature (22 ± 1°C). Embryos were exposed for a 24-hr period followed by either immediate evaluation or an additional 24 hr of standard in vitro culture (darkness, 37°C) and evaluation thereafter. Effects were assayed by cytophotometric measurement of the DNA content in Feulgen-stained cell nuclei and by cell number. The incidence of DNA aneuploid embryos and DNA aneuploid cell nuclei per embryo, as well as the average nuclear DNA content, was not significantly different between exposed embryos and controls. Both in vitro culture and reduced temperature caused a decrease in cell number. The temperature-induced cell number decrease was reversible within 24 hr after return to 37°C. These results demonstrate that physical factors associated with in vitro culture do not increase DNA ploidy abnormalities in cultured preimplantation embryos. Mol. Reprod. Dev. 50:30–34, 1998. © 1998 Wiley-Liss, Inc.     

Biological dosimetry: the potential use of radiation-induced apoptosis in human T-lymphocytes

An assay for biological dosimetry based on the induction of apoptosis in human T-lymphocytes is described. Radiation-induced apoptosis was assessed by flow cytometric identification of cells displaying apoptosis-associated DNA condensation. CD4 and CD8 T-lymphocytes were analysed. They were recognized on the basis of their cell-surface antigens. Four parameters were measured for both cell types: cell size, granularity, antigen immunofluorescence and DNA content. Apoptosis was quantified as the fraction of CD4-, or CD8-positive cells with a characteristic reduction of cell size and DNA content. At doses below 1 Gy, levels of radiation-induced apoptosis increased for up to 5 days after irradiation. Optimal dose discrimination was observed 4 days after irradiation, at which time the dose-response curves were linear, with a slope of 8% ± 0.5% per 0.1 Gy. In controlled, dose-response experiments the lowest dose level at which the radiation-induced apoptosis frequency was still significantly above control was 0.05 Gy. After 5 days post-irradiation incubation, intra- and interdonor variations were measured and found to be similar; thus, apoptotic levels depend more on the dose than on the donor. The results demonstrate the potential of this assay as a biological dosimeter. Received: 27 February 1997 / Accepted in revised form: 14 May 1997     

Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus 40. II. Changes in the distribution of DNA content during the reversible transition between macrophage and nonmacrophage states in the cultures of tsA640-transformed macrophages

Cultures of mouse macrophage cell lines transformed by wild-type or the tsA640 mutant of simian virus 40 (SV40) show a reversible phenotypic transition between the nonmacrophage (proliferating phase) and the macrophage (stationary phase) states (Takayama, 1980; Tanigawa et al., 1983). Distribution of DNA content in the cultures of the tsA640-transformed macrophage lines in the process of the phenotypic transition was determined by flow cytometry. Taking the mean DNA content of mouse peritoneal macrophages as 1 unit in the scale of fluorescence intensity in the flow cytogram, the transformed macrophages showed, at 33 degrees C, two peaks, one located around the 1.0-unit position (peak 1.0) and the other around the 1.6-unit position (peak 1.6), and a plateau distribution continuing to 3.2 units. Peak 1.0 was predominant in the stationary-phase culture, whereas peak 1.6 was predominant in the proliferating-phase culture. Almost the entire population of the strictly resting culture, which was obtained by culturing the stationary-phase culture for a further 5 days at nonpermissive temperature (39 degrees C), was phagocytic, and had accumulated at peak 1.0. Cells in peak 1.0 moved to peak 1.6 and to higher positions, after the strictly resting culture was sparsely reseeded and incubated at 33 degrees C. In contrast, the DNA content distribution of the successively proliferating cells, which were obtained by repeated passage of an extensively proliferating culture and none of which were phagocytic, was similar to that of proliferating hypotetraploid BALB/c3T3 fibroblasts with a G1 peak at 1.6 unit followed by a plateau containing S- and G2-phase cells. The peak 1.0 cell population appeared from the recloned population of the successively proliferating cells in company with the restoration of the culture condition-dependent phagocytic ability when cocultured with primary macrophages. Each peak in the flow cytogram reflected fairly well DNA content per cell as determined by other methods.     

The effect of dexamethasone on the cytotoxic and enzymatic response of cultured endothelial cells to radiation

Experiments were conducted to determine (1) whether glucocorticoids directly protected endothelial cells (EC) from radiation and (2) if angiotensin converting enzyme (ACE) activity, known to be increased by glucocorticoid, played a role in the EC response to radiation. Confluent monolayers of EC cultured from bovine aorta EC were treated with dexamethasone (10(-6) M); after irradiation (5.0 Gy, 60Co gamma), ACE and lactate dehydrogenase (LDH) activities, DNA and protein contents, and nuclei number were measured. Twenty-four hours after 5 Gy, there was increased cell loss (-40%, P less than 0.001), greater LDH release (greater than 100%, P less than 0.001), more LDH activity per cell (+40%, P less than 0.001), and unchanged ACE activity compared to sham-irradiated control EC. However, 48 hr after 5 Gy, ACE activity per cell was decreased (-24%, P less than 0.005). A 48-hr exposure to dexamethasone alone was accompanied by a slight cell loss (-10%, P less than 0.001) and increased cellular ACE activity (+40-140%, P less than 0.001), but a 24-hr dexamethasone exposure was not cytotoxic and did not change ACE activity. Dexamethasone exposure for 48 hr before and after irradiation did not attenuate cell loss or LDH release. However, combined dexamethasone treatment and radiation increased cellular ACE activity at a time when neither agent alone had an effect (24-hr dexamethasone exposure before 5 Gy and assayed 24 hr after 5 Gy). This interaction between radiation and dexamethasone treatment suggests that the glucocorticoid modifies the cell's response to injury. Although this interaction does not ameliorate radiation cytotoxicity, maintenance of ACE levels in injured vessels by hormones may have physiological significance in the hemodynamics of irradiated tissues.     

Inhibition of DNA methylation in Friend erythroleukemia cells by actinomycin D

Actinomycin D caused the production of hypomethylated DNA in cultured Friend erythroleukemia cells at cell culture concentrations of 1-4 ng per ml. Inhibition of DNA methyltransferase in cell-free assays was kinetically complex, with mixed-type inhibition. Cornish-Bowden graphical analysis was used to derive a Ki of about 35 nmol Act D per mg DNA. Although nuclei from drug-treated cells were found to contain hypomethylated DNA and DNA methyltransferase could be extracted from the nuclei, the methyl-accepting ability of DNA in whole nuclei themselves was not elevated. We conclude that the low level of Act D bound to DNA in the nuclei is sufficient to prevent the remethylation of hypomethylated sites.     

Intensive nuclear selection in tryptophan limited cultures of Escherichia coli B/r/l, trp

Summary The paradoxical constancy of the rate of mutation to resistance to bacteriophage T5 was observed long ago by Novick and Szilard. Mutation rates are independent of growth rate in tryptophan limited chemostat cultures, even though both average cell volume and DNA content increase with generation time. To examine nuclear selection in these multinucleate cells, cultures of E. coli B/r/l, trp were exposed briefly to acridine orange/visible light, and cells repackaged to uninucleate forms by shifting growth rates up to 0.5 divisions per hr. The kinetics of accumulation of mutant nuclei indicates that only a single master nucleus replicates in each cell, one of its progeny becomes the master nucleus during the following nuclear generation in the same cell, and both progeny become master nuclei if the cell divides. Autoradiographs showed that master nuclei are located at the ends of cells and that uninucleate cells are produced at each division, except possibly for a small fraction of aberrant divisions. This intensive selection during replication and inheritance of master nuclei provides an explanation for the constancy of cellular mutation rates, which is due to the constancy of the rate of DNA replication in these cultures and the (almost) complete selection for mutant nuclei in mutated cells.Work supported by the U. S. Atomic Energy Commission.     

Characterization of abnormal nuclei in renal proximal tubules after irradiation

The distribution and kinetics of proximal tubular cells with abnormally large nuclei, which were observed in irradiated mouse kidneys before any other obvious histological effects, were investigated. Six months after the administration of 13 or 15 Gy, little histopathological change was noted, in the kidneys of C3H mice; however, proliferation of proximal tubular cells was stimulated, and some of these cells had abnormally large nuclei. The relative DNA content of these large nuclei was measured with a quantitative image analysis system. Most of the large nuclear cells had more than diploid DNA content. The labeling index of the large nuclei was higher than that of unselected proximal tubular nuclei. These cells might be hyperploid cells that are dying after having gone through an abortive mitotic division. Examination and quantitation of these abnormal nuclei should be useful in elucidating the steps involved in cell loss in the proximal tubules after irradiation and as an assay for radiation damage to the kidney.     

Division and Formation of the Macronuclei of Keronopsis rubra*

The hypotrichous ciliate Keronopsis rubra has ～10 micronuclei and ～100 small macronuclei. DNA synthesis proceeds synchronously in all macronuclei in the 2nd half of the cell cycle which takes about 24 hr at room temperature. A G₂ phase is virtually absent, each nucleus dividing as soon as the replication band has passed over it. The micronuclear S phase falls within macronuclear G₁ and is followed by immediate division. Comparative cytophotometric measurements of Feulgen-stained preparations indicate that the DNA content of G₁ macronuclei is scattered widely in a skewed normal distribution, with a peak corresponding to the DNA content of a G₁ micronucleus. Measurements of dividing macronuclei indicate unequal distribution of DNA between daughter nuclei and lead to the conclusion that the units of assortment must be smaller than whole genomes unless the micronucleus is polyploid. After conjugation, a large macronuclear anlage with threads resembling split prophase chromosomes is formed. The threads condense and pass singly into the cytoplasm where they are thought to give rise to the numerous small macronuclei of the vegetative cells.     

Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green

Pyronin Y (PY) was used, in flow cytometric (FCM) systems, to estimate the RNA content per cell in formalin fixed EL4 leukosis tumor cells, enzyme dispersed R3327-G rat prostatic adenocarcinoma cells, mouse spleen cells stimulated with concanavalin A, and human peripheral blood lymphocytes stimulated with phytohemagglutinin. Preincubation of the cells with methyl green (MG) blocked PY binding to DNA such that the intracellular fluorescence from MG-PY was due primarily to its binding to RNA. Treatment of the cells with ribonuclease resulted in a 3- to 5-fold reduction in the fluorescence intensity of intracellular MG-PY. Mitogen stimulation of either mouse or human lymphocytes resulted in an increase in DNA (propidium iodide fluorescence) and RNA (MG-PY fluorescence) content per cell over resting levels. Further, the changes in stimulated human lymphocyte DNA and RNA contents following 24, 48, and 72 hr of cell culture were monitored. The results showed that RNA levels were significantly increased prior to that of DNA. Also, the effects of different cell cycle phase specific blocking agents on lymphocyte cell cycle traverse were investigated. We found that: a) actinomycin D inhibited the increases in cellular RNA and DNA; b) hydroxyurea inhibited the increases in cellular RNA were only slightly reduced; c) tritiated thymidine caused an accumulation of cells having high DNA and RNA contents; and d) Colcemid promoted an accumulation of cells having high DNA contents while causing a reduction of cells having high RNA contents. These results were nearly identical to reports by other investigators using the metachromatic dye acridine orange to quantitate RNA per cell. Thus, the MG-PY technique described is indicated to provide a stable and accurate measure of RNA content per cell.     

The radiosensitivity of the chromosomes of the cells of human squamous cell carcinoma cell lines.

Measurement of the radiation sensitivity of chromosomes was used to address the influence of cell cycle distribution and of DNA content and ploidy on radiation responses in seven human squamous cell carcinoma cell lines. The cell lines varied about twofold in DNA content and chromosome number, and the X-ray sensitivities (D0) of the lines ranged from 1.1 to 2.7 Gy. The more resistant cell lines (D0 greater than 1.8 Gy) had faster growth rates and larger proportions of cells in S phase in asynchronous cultures. Aberration frequencies were measured in cells irradiated in G1 and G2 phase. The more resistant lines had fewer induced aberrations in both phases than did sensitive lines, implying that they were more resistant to radiation in both of these cell cycle phases. Therefore, while the larger S-phase population seen in the resistant cell lines probably contributes to the resistant phenotype, it cannot explain all of the intrinsic differences in radiation sensitivity. There was no relationship between DNA content and radiation sensitivity as measured by the cell survival assay or the induction of chromosome aberrations, although cells with larger DNA contents tended to have more chromosome damage per cell at equitoxic doses.     

Kinetics of long-term (72 hr) calcium content during mitogen activation of cultured human T lymphocytes

In vitro stimulation of human blood lymphocytes with mitogen resulted in an increased intracellular content of Ca2+ per unit cell volume. This increase in Ca2+ content of lectin-activated cells reached a maximum after 24 hr of culture and thereafter slowly declined. Brief treatment of cells at 24 hr of culture with the Ca2+ ionophore A23187 in combination with EGTA resulted in a larger release of Ca2+ from cells in mitogen-stimulated cultures than from cells in control cultures. This indicates that the Ca2+ is accumulated intracellularly but is readily exchangeable. At 24 hr of culture the increase in cellular Ca2+ correlated well with the proliferative response as measured by 3H-thymidine incorporation. Ca2+ influx at 24 and 48 hr of culture was markedly enhanced in the mitogenically stimulated cells as compared either to cells cultured for 1 and 72 hr or cells cultured without mitogen.     

Synthesis of nucleic acids in the Schwann cells as the early cellular response to nerve injury

Abstract— The early effect of mechanical injury of the sciatic nerve in cats and rats on the amount of nucleic acids and on the number of Schwann cells was studied. The content of RNA and DNA increased at both ends of a severed nerve as early as 2 hr after operation; at 8 hr and up to 24 hr, their level was about twice as high as at the beginning of experiment. The increase in nucleic acids seemed to be chiefly due to their biosynthesis in the Schwann cells although the possibility of a contribution of axonal RNA must also be taken into consideration. The division of Schwann cell nuclei was observed not earlier than 36 hr after operation. The injury stimulates the synthesis of nucleic acids not only in the Schwann cells directly damaged, but also in those from neighbouring internodes. The increase in DNA and RNA observed 24 hr after a fresh lesion had been made in a degenerating nerve was smaller than the increase observed 24 hr after cutting or crushing a normal nerve.     

AN ANALYSIS OF DNA LOSS AND SYNTHESIS IN THE RAT ADRENAL MEDULLA NUCLEI UPON COLD STIMULATION

The peculiar changes previously observed in DNA content of rat adrenal medulla cell nuclei upon intermittent cold exposure (15 hr at +4°C followed by 9 hr at room temperature) have been further studied with the aid of Feulgen histophotometry and H₃-thymidine radioautography. The amount of DNA decreases progressively with increasing length of cold exposure until 300 hr (-32%). Later a rapid change takes place, whereby DNA content per nucleus returns to values which are slightly, but consistently lower than normal. At termination of a period of cumulative exposure to cold, an analysis of a whole-day experimental cycle shows that the DNA decrease is due to loss of DNA during cold exposure and that DNA synthesis occurs upon return to room temperature. The balance between these two processes can be divided into three stages: (a) loss of DNA up to 300 hr of cumulative cold exposure; (b) marked increase in DNA by 350 hr; (c) oscillation around zero or slightly negative at 400 hr and beyond. These variations are due to: (1) the extension of DNA synthesis into the period of cold exposure as clearly demonstrated by radioautography (stage b), and (2) a later still greater DNA loss (stage c) which partly offsets the increased synthesis. A complex pattern of adaptation of the adrenal medulla cells, as regards DNA content, to the repetitive cold stimulus is thus demonstrated.     

Dose-rate effects in mammalian cells. IV. Repairable and nonrepairable damage in noncycling C3H 10T 1/2 cells

Repairable and nonrepairable components of gamma-ray damage leading to cell reproductive death were determined by measuring the range over which dose rate influenced the response of non-cycling C3H 10T 1/2 mouse cells. Cell proliferation and cell cycle redistribution were eliminated as factors influencing the dose-rate effect in the system by irradiating confluent monolayers of contact inhibited cells. The radiosensitivity of the cells did not change, and no selective loss of damaged cells occurred over the extended treatment times. A pronounced dose-rate effect was observed over the range between 55.6 and 0.29 Gy/hr, but a limit to the repair-dependent dose-rate effect was reached at 0.29 Gy/hr since no further reduction in effect per unit dose was observed when the dose rate was reduced to 0.17 or 0.06 Gy/hr. The survival curves, which were simple exponential functions of dose at dose rates of 0.29 Gy/hr and below, have a common Do of 7.32 Gy and represent an accurate measurement of the nonrepairable component of damage. Log-phase cultures showed remarkably different responses over the range of dose rates, due in large part to cell cycle redistribution and in some cases, cell proliferation during exposures. The results of these studies were compared with time-dose relationships used in clinical brachy-therapy and agree remarkably well with corrections in total dose suggested by R. Paterson [Br. J. Radiol. 25, 505-516 (1952)] and A.E.S. Green [cited in F. Ellis, Curr. Top. Radiat. Res. Q. 4, 357-397 (1968)] when the standard treatment time is changed. Comparison of our data with in vivo isoeffect curves of total dose vs dose per fraction for "early" and "late" tissue responses indicate that cell cycle redistribution should not be ignored as a factor influencing time-dose relationships in radiotherapy.     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.