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.     

Dissociation by cytochalasin B of movement, DNA synthesis and transport in 3T3 cells.

Cytochalasin B was used as a tool to study the inter-relationships between cell movement, the reinitiated DNA synthesis and the enhanced transport of specific small molecules stimulated by serum in quiescent 3T3 cells. Cytochalasin at concentrations of less than 1 mug/ml inhibits serum-stimulated movement within the monolayer and migration into a wound. Even at ten times this concentration there is little effect on the increase in DNA in the culture, indicating that movement away from neighboring cells is not required for the initiation of DNA synthesis. While DNA synthesis is not inhibited by concentrations of cytochalasin up to 10 mug/ml, the increased thymidine transport which is associated with the onset of the S phase of the cell cycle is inhibited and DNA synthesis cannot be measured by the labelling of nuclei with radioactive thymidine. Cytochalasin has a differential effect on the early transport changes produced by serum addition. Glucose transport is inhibited by low concentrations of the drug (less than 1 mug/ml) while the enhanced uptake of phosphate and uridine is unaffected by a 10-fold increase in concentration. Although the doses of cytochalasin required for 50% inhibition of hexose uptake and of cell movement are the same, no causal relationship between sugar transport and locomotion can be demonstrated. Cytochalasin affects membrane functions in at least two different ways. The drug inhibits the uptake of glucose directly but affects only the S-phase associated increase in thymidine transport.     

AUTORADIOGRAPHIC DETECTION OF DNA POLYMERASE CONTAINING NUCLEI IN SARCOMA 180 ASCITES CELLS

A technique has been developed allowing the autoradiographic detection of incorporation of ³H-thymidine-5'-triphosphate (³H-TTP) into nuclear DNA of smears of Sarcoma-180 (S-180) mouse ascites tumors under the direction of the cell's own nuclear DNA polymerase. Dried smears are dipped into an agar solution, which strips cytoplasm from the nuclei, and are then air dried and incubated with a buffered mixture containing four nucleotide triphosphates (one labeled), Mg⁺⁺, and Ficoll, with the cell's own DNA acting as primer. The incorporation of ³H-TTP into the nuclei, like the cell free DNA polymerase assay, is largely dependent on the presence of all four nucleotide triphosphates and Mg⁺⁺ and produces a product which is DNase sensitive and RNase resistant.
DNA polymerase activity, as studied in a cell free assay, decreases with tumor age. This correlates well with a decreasing ³H-TTP labeling index in autoradiographs of aging tumors. The ³H-TTP labeling index has also been shown to exceed but parallel the in vivo ³H-thymidine (³H-TdR) pulse labeling index for all tumor ages examined.
In at least some cell systems DNA polymerase seems characteristic of cells in cycle. The autoradiographic detection of nuclei containing the enzyme offers a new tool for the study of tumor cytokinetics.     

Adaptation of the body to the effects of stress increases the resistance of cardiac nuclei to damaging effects of single-chain exogenous DNA

The effect of adaptation to stress exposure on the resistance of nuclear DNA to damaging effect of one-chain exogenous DNA was investigated by flow cytometric fluorescence method. It was shown that after the administration of one-chain DNA in concentration of 50 micrograms/ml into the nuclei suspension almost half of nuclear DNA was damaged in control. In adaptation this phenomenon was 5.5 times less pronounced. When the one-chain DNA concentration was increased, this protective effect of adaptation remained. The possible mechanism of the DNA-protective effect of adaptation is under consideration.     

INTERACTION OF POLY-L-LYSINE WITH CHROMATIN : Inhibition of In Situ RNA Synthesis Mediated by Escherichia coli RNA Polymerase

RNA polymerase from Escherichia coli was used in conjunction with labeled nucleosides as an autoradiographic reagent to study the availability of template in the chromatin of fixed nuclei and chromosomes Sequential treatments of the tissues with acid and poly-L-lysine were used to compare the effect of these treatments on the availability of template with the previously reported effects on the in situ priming for Escherichia coli DNA polymerase Acid treatment was found to increase the in situ activity of both enzymes, while poly-L-lysine strongly inhibited the in situ reactions mediated by RNA and DNA polymerases. When the DNA polymerase reaction was previously carried out on alcohol-fixed chicken blood smears, leukocyte nuclei primed extensively for DNA synthesis. In contrast, we did not detect incorporation into intact nuclei of any cell type in alcohol-fixed blood smears that were treated with RNA polymerase.     

Degradation of HeLa cell chromatin by neocarzinostatin and its chromophore

Chromatin is the in vivo target site for neocarzinostatin, a DNA strand scission antitumor drug. The effect of neocarzinostatin and its active chromophore component on HeLa cell chromatin is described here. Chromatin consisting of a mixture of mono-, di-, tri- and larger nucleosome fragments is prepared by micrococcal nuclease digestion of HeLa cell nuclei. Drug-induced conversion of chromatin to smaller sized fragments is measured by electrophoresis of the DNA on non-denaturing 4% polyacrylamide gels. Chromatin breakdown measured under these conditions is double-stranded in nature. In the presence of 2 mM dithiothreitol, neocarzinostatin causes degradation of large chromatin fragments and a loss of distinct nucleosome peaks. Detection of chromatin breakdown by neocarzinostatin is dependent upon the concentration of chromatin in the assay. When chromatin is increased from 14 to 70 micrograms/ml, changes in the larger fragments caused by 100 micrograms/ml neocarzinostatin become less obvious are are almost undetectable at 140 micrograms/ml chromatin. No change is observed when chromatin is treated with either neocarzinostatin or its chromophore in the absence of dithiothreitol. For detectable levels of chromatin degradation, 10 micrograms/ml neocarzinostatin is required compared to only 2.5 microgram/ml chromosome (expressed in microgram equivalent neocarzinostatin). Such degradation also occurs more rapidly with chromophore than with neocarzinostatin. Digestion of chromatin with neocarzinostatin continues for at least 30 min at 37 degrees C, while similar degradation caused by chromophore is complete in 1 min. Neocarzinostatin levels which actively degrade isolated chromatin can also effect release of soluble chromatin from intact nuclei. The released chromatin can serve as a substrate for micrococcal nuclease digestion. Such chromatin studies should prove useful in characterizing the mechanism of action of DNA reactive drugs such as neocarzinostatin.     

Overall changes in chromatin sensitivity to DNase I during differentiation

The DNase I sensitivity of total chromatin was studied in fixed cells and nuclei isolated from proliferating and terminally differentiated cells, by measuring the incorporation of labelled nucleotides into DNase-sensitive sites, and electrophoresis of DNA isolated from DNase-treated nuclei. The unfixed nuclei were sensitive to digestion at around 10 micrograms/ml, the fixed cells at 30 ng/ml DNase I concentration. Proliferating Rauscher leukemia cells were more digestible than normal spleen cells. The DNase I sensitivity of the human HL60 leukemia line decreased upon DMSO-induced differentiation but still exceeded the digestibility of nuclei from normal human peripheral blood. A novel flow-cytometric technique was developed to study DNase sensitivity at the cell level. It confirmed the relative resistance of differentiated cells to DNase I and ruled out the possibility that this could be due to an altered distribution of cell cycle phases. The overall DNase I sensitivity of chromatin was compared with the sensitivity of the c-myc gene and the myc-associated hypersensitive sites. The latter sites were detected at 1 microgram/ml DNase I in HL60 nuclei. They disappeared partially upon DMSO-induced differentiation. At 10 micrograms/ml, myc was degraded in both growing and differentiating HL60, but not in HPB cells. These data suggest that a progressive condensation of the chromatin occurs during terminal differentiation which gradually involves specific genes that need to be inactivated.     

Cytoflurometric nuclear DNA-determinations in infant,adolescent, adult and aging human hearts

Summary The progress of polyploidization in the human heart muscle cell was investigated by cytofluorometry, involving selective measurements of heart muscle cell nuclei. Thirty-two tissue samples, taken from the free wall of the left ventricle of each autopsied heart, were fixed in Carnoy's fluid. From thick (100–150 m) paraffin sections, isolated cells for smears were obtained by enzyme digestion and ultrasonic treatment. The smears were stained with azocarmin G to eliminate background fluorescence and subsequently stained by an acriflavine-Feulgen reaction. Cytofluorometric DNA-determinations were carried out selectively on heart muscle cell nuclei, using the muscle striations revealed by azocarmin G-fluorescence as specific markers. The dynamic process of polyploidization in normal hearts could be divided into four stages. In the first stage (under 1 year of age), almost all heart muscle cell nuclei (94.3±1.8%) were diploid. In the second stage (1 to 9 years of age), the number of tetraploid nuclei increased (13.6±7.1%). In the third stage (9 to 22 years of age), octaploid nuclei first appeared and the number of tetraploid nuclei increased (26.7±3.9%). The DNA pattern in the fourth stage (22 to 75 years of age) was relatively constant, with a ratio of diploid (62.4±8.7%), tetraploid (31.4±6.7%) and octaploid (5.8±3.9%) nuclei. From these results it was concluded that physiological polyploidization progresses in proportion to the increase of heart weight. The frequency of polyploid nuclei in human heart was not so high as resported by previous investigators.     

Fluorescence decay measurements for determining the relative content of ethidium bromide to DNA in situ in cell nuclei

A fluorometric method for the determination of the amount of ethidium bromide (EB) bound to DNA in situ in cell nuclei is discussed. Even when the EB content was very small, the molar ratio of DNA-phosphorus (DNA-p) to dye (P/D ratio) could be estimated by measuring the lifetime of the transient fluorescence of the EB-DNA complex as a function of the P/D ratio. To examine the relationship between the fluorescence intensity, lifetime, and P/D ratio, polyacrylamide gel film containing 4.7 mM DNA-p was used as a model DNA tissue, and its fluorescence was measured using a nanosecond microfluorometer. The fluorescence intensity showed a maximum at P/D = 6. The fluorescence lifetime increased with the P/D ratio, and this was accompanied by a proportional increase in the quantum efficiency. Thus, the lifetime value was an effective parameter for the determination of the P/D ratio in situ in tissue. When this approach was applied to tissue sections of mouse liver treated with solutions of EB at concentrations of 10 and 50 micrograms/ml, the fluorescence lifetimes on cell nuclei were 18.9 and 17.4 ns with P/D ratios of 20 and 12, respectively, as based on the model-tissue experiments. When the P/D ratio was 20, the concentration of EB in the nucleus was approximately 1.5 mM, i.e., 60 times higher than that in the staining solution.     

Influence of smear preparation and fixatives on the DNA ploidy and the morphonuclear features of the MXT mammary tumor and normal tissues in the mouse

We compared cytomorphonuclear parameters--related to DNA histogram and chromatin distribution--on MXT mouse mammary tumor and murine normal cells from fresh squash smears or from deparaffinized tissue smears fixed in several fluids. We used the SAMBA 200 cell image processor with software allowing for the discrimination of parameters computed on Feulgen-stained nuclei. The spectrophotometric results--assessed by integrated optical density values--indicate that the nuclei from deparaffinized tissue fixed in Bouin's fluid are around 50% less stained than those fixed in formalin or ethanol-formalin-acetic acid (EFA). The fresh smears of nuclei fixed in formalin contain a less well-defined and more homogeneous chromatin than after Bouin's or EFA fixation. This has led to the conclusion that morphonuclear parameter comparisons performed on tissues differently processed or from different origins present severe limitations.     

Fluorescence decay measurements for determining the relative content of ethidium bromide to DNA in situ in cell nuclei

Summary A fluorometric method for the determination of the amount of ethidium bromide (EB) bound to DNA in situ in cell nuclei is discussed. Even when the EB content was very small, the molar ratio of DNA-phosphorus (DNA-p) to dye (P/D ratio) could be estimated by measuring the lifetime of the transient fluorescence of the EB-DNA complex as a function of the P/D ratio. To examine the relationship between the fluorescence intensity, lifetime, and P/D ratio, polyacrylamide gel film containing 4.7 mM DNA-P was used as a model DNA tissue, and its fluorescence was measured using a nanosecond microfluorometer. The fluorescence intensity showed a maximum at P/D=6. The fluorescence lifetime increased with the P/D ratio, and this was accompanied by a proportional increase in the quantum efficiency. Thus, the lifetime value was an effective parameter for the determination of the P/D ratio in situ in tissue. When this approach was applied to tissue sections of mouse liver treated with solutions of EB at concentrations of 10 and 50 g/ml, the fluorescence lifetimes on cell nuclei were 18.9 and 17.4 ns with P/D ratios of 20 and 12, respectively, as based on the model-tissue experiments. When the P/D ratio was 20, the concentration of EB in the nucleus was approximately 1.5 mM, i.e., 60 times higher than that in the staining solution.     

Effect of Ehrlich ascites cell chalone on nascent DNA synthesis in isolated nuclei

Ehrlich Ascites Tumor (EAT) chalone has been shown to inhibit nascent DNA synthesis by inhibiting DNA polymerase alpha and beta (Nakai, 1976), but one of the problems in studying eurkaryotic DNA replication has been the relative impermeability of the cell membrane to precursors and macromolecules; hence, to circumvent this restriction without sacrificing the integrity of the replication process, a broken cell system utilizing nuclei in aqueous media was investigated. Isolated nuclei appear to continue the process of DNA replication that was proceeding in vivo before their isolation and under optimal concitions are able to initiate new synthesis (Fraser & Huberman, 1977). The effects of partially purified EAT chalone on nascent DNA could be studied directly in this nuclear system, which excluded effects of the cell membrane, nucleotide pools and other cytosol elements. A concentration-related inhibition of [3H]thymidine triphosphate ([3H]dTTP) incorporation was noted over a chalone range of 50-200 micrograms/ml. It appears that chalone can inhibit DNA polymerase alpha directly within the nucleus without an intermediate step such as a cell membrane receptor.     

Influence of different cell extraction methods on cytometric features.

The purpose of this study was to investigate the influence of different cell extraction procedures on relative nuclear DNA content (IOD), nuclear area, and texture features of Feulgen-stained nuclei. In imprints and smears of fine-needle aspirates and suspensions from one human liver specimen, 50 diploid, 50 tetraploid, and 25 octaploid nuclei were measured from each slide. In addition, for DNA measurements, the progressive mean of IOD and tetraploid/diploid and octaploid/diploid ratios was calculated. The results show that the progressive mean of the IOD is constant after measuring 25-30 nuclei. For the three types of specimens, the IOD of diploid nuclei varied slightly. The average coefficient of variation was about 5% for the fine-needle aspirates, imprints, and suspensions. For all tissue sampling methods, the 99% confidence limits of the tetraploid/diploid ratio and octaploid/diploid ratio were within the range of 1.9-2.1 and 3.7-4.3, respectively. The nuclear area and most of the texture features showed a significant difference (p less than 0.01) between the three sampling methods in all nuclear populations. In conclusion, different tissue sampling methods have little or no effect on DNA-related IOD measurements, whereas the outcome of nuclear area and texture features is very dependent of the cell extraction procedure.     

Chromatin Fluorescence after Carmine Staining

After staining with dilute solutions (0.1 mg/ml in distilled water) of commercial carmine, a strong reddish orange fluorescence was observed in nuclei from cell smears and frozen and paraffin tissue sections. Optimal exciting light ws 436 nm (violet-blue) or 450-490 nm (blue). Compact chromatin from interphase nuclei, mitotic and meiotic chromosomes and the kinetoplast of Trypanosoma cruzi showed the highest fluorescence, while the basophilic cytoplasm appeared weakly fluorescent. No emission was observed in cartilage matrix, mast cell granules or goblet cell mucin. This selective method could be valuable in microscopic and cytochemical studies on chromatin because the carmine fluorescence is stable and preparations can be dehydrated and mounted permanently without changes in the fluorescence pattern.     

Chromatin fluorescence after carmine staining

After staining with dilute solutions (0.1 mg/ml in distilled water) of commercial carmine, a strong reddish orange fluorescence was observed in nuclei from cell smears and frozen and paraffin tissue sections. Optimal exciting light was 436 nm (violet-blue) or 450-490 nm (blue). Compact chromatin from interphase nuclei, mitotic and meiotic chromosomes and the kinetoplast of Trypanosoma cruzi showed the highest fluorescence, while the basophilic cytoplasm appeared weakly fluorescent. No emission was observed in cartilage matrix, mast cell granules or goblet cell mucin. This selective method could be valuable in microscopic and cytochemical studies on chromatin because the carmine fluorescence is stable and preparations can be dehydrated and mounted permanently without changes in the fluorescence pattern.     

EFFECT OF EHRLICH ASCITES CELL CHALONE ON NASCENT DNA SYNTHESIS IN ISOLATED NUCLEI

Ehrlich Ascites Tumor (EAT) chalone has been shown to inhibit nascent DNA synthesis by inhibiting DNA polymerase α and β (Nakai, 1976), but one of the problems in studying eurkaryotic DNA replication has been the relative impermeability of the cell membrane to precursors and macromolecules; hence, to circumvent this restriction without sacrificing the integrity of the replication process, a broken cell system utilizing nuclei in aqueous media was investigated. Isolated nuclei appear to continue the process of DNA replication that was proceeding in vivo before their isolation and under optimal conditions are able to initiate new synthesis (Fraser & Huberman, 1977). The effects of partially purified EAT chalone on nascent DNA could be studied directly in this nuclear system, which excluded effects of the cell membrane, nucleotide pools and other cytosol elements. A concentration-related inhibition of [³H]thymidine triphosphate ([³H]-dTTP) incorporation was noted over a chalone range of 50–200 μg/ml. It appears that chalone can inhibit DNA polymerase α directly within the nucleus without an intermediate step such as a cell membrane receptor.     

Protein synthesis inhibitors, like growth factors, may render resting 3T3 cells competent for DNA synthesis: a radioautographic and cell fusion study

Serum-deprived (0.1-0.2%) resting NIH 3T3 mouse fibroblasts pre-incubated with cycloheximide (7.5 micrograms/ml), or puromycin (10 micrograms/ml), were fused with stimulated cells taken 10 h after changing the medium to one containing 10% serum, and DNA synthesis was investigated in the nuclei of monokaryons, homodikaryons and heterodikaryons using radioautography with the double-labelling technique. Pre-incubation of resting cells with inhibitors of protein synthesis for 1-4 h abolished their ability to suppress DNA synthesis in stimulated nuclei in heterokaryons. Three hours after the removal of cycloheximide from the medium, the resting cells acquired once again the inhibitory capacity for entry of stimulated nuclei into the S period. This inhibitory influence disappeared also in the case of post-fusion cycloheximide application as well as following an 8-12 h pre-treatment of resting cells with actinomycin D (1 microgram/ml) prior to fusion. Pre-incubation of resting cells for 12 h with PDGF (1 u/ml-1) followed by an 8-48 h incubation in serum-free medium stimulated the onset of DNA synthesis. A brief exposure (45 min) of resting cells to cycloheximide (7.5 micrograms/ml), or puromycin (7.5 micrograms/ml), exerted a similar effect, inducing by itself the entry of cells into the S period. The results support the assumption that acquirement, by resting cells, of competence for DNA replication includes as a necessary step the down-regulation of intracellular growth inhibitors whose formation depends on protein synthesis.     

Expression of cell-cycle-dependent genes in serum stimulated cells whose entry into S phase is blocked by cytochalasin D

A low concentration (0.6 micrograms/ml) of cytochalasin D inhibits the initiation of DNA synthesis after serum stimulation of growth-arrested GC-7 cells. Since actin-containing structures are suggested to be involved in the transfer of the growth signal to nuclei and in the synthesis and transport of nascent RNA, the effect of cytochalasin D on the expression of cell-cycle-regulated genes after serum stimulation was studied by Northern blot analysis. Cytoplasmic accumulation of such mRNAs as or c-fos, c-myc, beta-actin an ornithine decarboxylase occurred in serum-stimulated cells regardless of the presence of cytochalasin D, whereas that of thymidine kinase and histone H3 was blocked by the drug.