Flow cytometric DNA analysis on fine needle aspiration biopsies of liver lesions

Flow cytometric DNA analysis on fine needle aspiration biopsies of liver lesions The DNA cell content of 39 fine needle aspiration biopsies (FNAs) from five benign liver lesions, nine hepatocellular carcinomas (HCCs), and 25 metastatic tumours was analysed in a prospective fashion by flow cytometry (FCM). All benign lesions were diploid. Aneuploidy was found in five (55.6%) HCCs and in nine (36%) metastatic tumours. DNA index (DI) differences were not significant. The S-phase fraction (SPF) was higher in the malignant tumours, both combined (P < 0.02) and separated primary and metastatic (P < 0.05). We could not demonstrate an association between diploidy and percentage of benign hepatocytes in the smears of malignant tumours. The serum alpha-fetoprotein (AFP) level did not correlate with ploidy, DI, or SPF in the HCCs. In conclusion, ploidy and DI do not discriminate between benign and malignant liver lesions, but the SPF is higher in malignant tumours. DNA analysis does not help to distinguish primary from metastatic liver tumours. The presence of benign hepatocytes in samples from malignant tumours does not seem to influence the analysis of ploidy by FCM.     

DNA flow cytometry of endoscopically examined colorectal adenomas and adenocarcinomas

DNA ploidy of 64 colorectal adenomas and 49 adenocarcinomas, examined endoscopically, was studied by flow cytometry. We found DNA aneuploidy in none of the 105 normal mucosa samples (0%), in 20 adenomas (31%), and in 36 adenocarcinomas (74%). DNA ploidy of adenomas correlated with size (P = 0.02) and degree of dysplasia (P less than 0.01) but not with histologic type. Adenomas had a 45% incidence of DNA aneuploid stem lines in the DNA index range of 0.80-1.20, compared with 8% in the case of adenocarcinomas. The distribution of the DNA index values of adenocarcinomas was approximately normal, with a mean value 1.63 +/- 0.28. The mean DNA index for the three cases of "carcinoma in adenoma" with invasion of the stalk of the adenoma was 1.52 +/- 0.18. These results, using DNA flow cytometry, provide evidence for the progression of colorectal adenoma to adenocarcinoma. The classification of adenomas according to DNA ploidy may be information of considerable practical value to the clinician in predicting risk of further adenomas and/or risk of cancer.     

Ultramicrochemical determination of nucleic acids in individual cells using the Zeiss UMSP-I microspectrophotometer. Application to isolated rat hepatocytes of different ploidy classes

Synopsis Edström's method for the ultramicrochemical determination of RNA and DNA in individual cells was modified for the measurement of extinction in u.v. light with the aid of the Zeiss scanning microspectrophotometer UMSP-I. With this new procedure, nucleic acids down to about 3 pg RNA or about 4 pg DNA can be measured with a very high accuracy.The method was applied to enzymatically isolated rat liver parenchymal cells. A mean DNA content of 6.52 pg was found for diploid cells. The DNA content of mononuclear cells of different ploidy levels and of binuclear cells showed a close proportionality with the nuclear ploidy and the number of nuclei per cell. The RNA content of mononuclear diploid cells amounted to 33.4 pg, yielding an RNA/DNA ratio of 5.12. The RNA/DNA ratio was similar for binuclear and mononuclear cells of the same ploidy level but decreased considerably with increasing nuclear ploidy.     

Regeneration of the liver of Chinese hamster Cricetulus griseus

Using cytofluorimetry and interferometry, hepatocyte DNA, dry mass and distribution of hepatocyte ploidy classes were measured in hamsters Cricetulus griseus in 1 month after partial hepatoctomy. Ploidy of normal liver hepatocyte was 2.35 +/- 0.03 (mean +/- SD) c. Modal ploidy class was presented by mononuclear hepatocytes with diploid nuclei (82.4 +/- 1.3 %). Hepatocyte dry mass was 605.2 +/- 4.8 pg. One month after partial hepatectomy the distribution of ploidy classes and dry mass of hepatocyte did not change. A similar hepatectomy in mice resulted in significant polyploidization of liver parenchyma: the middle level of hepatocyte ploidy increased by 32% and mononuclear octaploid cells, the number of which increased 5-fold, composed modal ploidy class in place of 4cx2-hepatocytes predominated in control mice. The number of 8cx2-hepatocytes in the liver of mice creased by more than 5-fold. Thus, in contrast with mice, in hamsters Cricetulus griseus an increase in the liver mass followed partial hepatectomy depended completely on hepatocyte proliferation.     

Effects of Polyploidy on Photosynthetic Rates, Photosynthetic Enzymes, Contents of DNA, Chlorophyll, and Sizes and Numbers of Photosynthetic Cells in the C(4) Dicot Atriplex confertifolia

Photosynthetic rates, chlorophyll content, and activities of several photosynthetic enzymes were determined per cell, per unit DNA, and per unit leaf area in five ploidal levels of the C₄ dicot Atriplex confertifolia. Volumes of bundle sheath and mesophyll protoplasts were measured in enzymatic digestions of leaf tissue. Photosynthetic rates per cell, contents of DNA per cell, and activities of the bundle sheath enzymes ribulose 1,5-bisphosphate carboxylase (RuBPC) and NAD-malic enzyme per cell were correlated with ploidal level at 99% or 95% confidence levels, and the results suggested a near proportional relationship between gene dosage and gene products. There was also a high correlation between volume of mesophyll and bundle sheath cells and the ploidal level. Contents of DNA per cell, activity of RuBPC per cell, and volumes of cells were correlated with photosynthetic rate per cell at the 95% confidence level. The mesophyll cells did not respond to changes in ploidy like the bundle sheath cells. In the mesophyll cells the chlorophyll content per cell was constant at different ploidal levels, there was less increase in cell volume than in bundle sheath cells with an increase in ploidy, and there was not a significant correlation (at 95% level) of phosphoenolpyruvate carboxylase activity or content and pyruvate,Pi dikinase activity with increase in ploidy. The number of photosynthetic cells per unit leaf area progressively decreased with increasing ploidy from diploid to hexaploid, but thereafter remained constant in octaploid and decaploid plants. Numbers of cells per leaf area were not correlated with cell volumes. The mean photosynthetic rates per unit leaf area were lowest in the diploid, similar in 4×, 6×, and 8×, and highest in the decaploid. The photosynthetic rate per leaf area was highly correlated with the DNA content per leaf area.     

Cellular mechanisms of cirrhotic rat liver regeneration. II. Proliferation, polyploidization and hypertrophy after partial hepatectomy

Using cytofluorimetry and absorptional cytophotometry, hepatocyte DNA and total protein contents were measured in intact and cirrhotic rats in 1, 3 and 6 months after partial hepatectomy (PH). It has been found that within one month of intact rat liver regeneration the level of hepatocyte ploidy rised by 25% to remain elevated for the next 6 months. This was due mainly to reducing the number of cells with diploid nuclei (2c 2-fold, 2c x 2 - 6.6-fold) and to rising the number of octaploid hepatocytes. In cirrhotic animals the ploidy level in hepatocytes increased in 3 months after PH, and decreased by 15% in 6 months. The number of hepatocytes with diploid nuclei (2c and 2c x 2) increased within 3-6 months in both control and cirrhotic rats. The protein content per diploid hepatocyte rised by 30% within 3-6 months of liver regeneration after PH. Special calculations have shown that within 3 months after PH the increase in the liver mass of control and cirrhotic rats was due completely to hepatocyte DNA synthesis, i. e. proliferation and polyploidization. Within the next 3 months of liver regeneration after PH, the contribution of polyploidization to liver mass increase was negative because of depolyploidization of liver parenchyma cell population. At this time hypertrophy was the main process determining the liver mass increase.     

Development of binuclearity and DNA-polyploidization in the growing mouse liver

Summary Suspensions of intact liver cells were prepared from 36 male NMRI mice of different age after perfusion of the liver with ice-cold calcium- and magnesium-free phosphate buffer (CMF). The suspensions of the isolated hepatocytes were smeared on slides, fixed, hydrolized and stained by fluorescent acriflavine-Schiff-Feulgen reaction. The number of nuclei per cell was determined in a phase-contrast microscope. Quantitative fluorescent cytophotometric measurements of nuclear Feulgen-DNA were performed on individual nuclei. At the age of 0.5 month, 55% of the hepatocytes were found to be mononuclear, 45% binuclear. In the animal groups aged 1 month, 1.5 months, 3 months, 6 months and 12 months, the percentage of binuclear hepatocytes remained constant at about 80%. Very few liver cells with 3 or 4 nuclei were detected. Feulgen-DNA-measurements revealed a predominance of 2c and 4c nuclei at ages 1 month and 1.5 months with logarithmic increase of 8c nuclei and a decrease of the 2c nuclei. From 1.5 months on 16c nuclei were found, which never exceeded 8%. When total DNA-ploidy of the hepatocytes was calculated similar kinetics at a higher ploidy level were observed. 2c hepatocytes existed in small percentages at very young ages up to 1.5 months, but were also occasionally found in older animals. With increasing age the number of 16c hepatocytes increased logarithmically with a concomitant decrease of the 4c hepatocytes. The percentage of 8c liver cells remained more or less constant. Few hepatocytes with a 32c total DNA content were found in mice aged 3 months and older. In one-year-old mice the mean DNA-ploidy was calculated to be 5.8c per liver nucleus and 10.0c per whole hepatocyte.Supported by Deutsche Forschungsgemeinschaft, Grant No Bo 395/5     

Changes of ploidy during the Azotobacter vinelandii growth cycle.

The size of the Azotobacter vinelandii chromosome is approximately 4,700 kb, as calculated by pulsed-field electrophoretic separation of fragments digested with the rarely cutting endonucleases SpeI and SwaI. Surveys of DNA content per cell by flow cytometry indicated the existence of ploidy changes during the A. vinelandii growth cycle in rich medium. Early-exponential-phase cells have a ploidy level similar to that of Escherichia coli or Salmonella typhimurium (probably ca. four chromosomes per cell), but a continuous increase of DNA content per cell is observed during growth. Late-exponential-phase cells may contain > 40 chromosomes per cell, while cells in the early stationary stage may contain > 80 chromosomes per cell. In late-stationary-phase cultures, the DNA content per cell is even higher, probably over 100 chromosome equivalents per cell. A dramatic change is observed in old stationary-phase cultures, when the population of highly polyploid bacteria segregates cells with low ploidy. The DNA content of the latter cells resembles that of cysts, suggesting that the process may reflect the onset of cyst differentiation. Cells with low ploidy are also formed when old stationary-phase cultures are diluted into fresh medium. Addition of rifampin to exponential-phase cultures causes a rapid increase in DNA content, indicating that A. vinelandii initiates multiple rounds of chromosome replication per cell division. Growth in minimal medium does not result in the spectacular changes of ploidy observed during rapid growth; this observation suggests that the polyploidy of A. vinelandii may not exist outside the laboratory.     

Differences in the flow and absorption cytometric DNA distributions of mouse hepatocytes and tumor cells

We have determined the DNA content, the ploidy levels, and the percentages of different cell types present in small and large mouse mammary tumors as well as in young and old mouse livers by using absorption and flow cytometry. Absorption cytometry data indicated a significant increase in the proportion of transformed G0/G1 cells in the tumors as compared to that of the stromal G0/G1 cells with progressive tumor growth. This increase was not detected by flow cytometry. In both young and old mouse livers, a small number of cells of higher ploidy (8C and 16C) were detected by absorption cytometry but were not apparent in histograms obtained by flow cytometry. Furthermore, changes in the proportions of liver cells of different ploidy with age were apparent in absorption cytometry data but not in flow cytometry data. In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.     

Effect of hydrocortisone on methylation and molecular population of DNA in rat liver]

The content of 5-methyl cytosine in rat liver DNA increases 1,7-fold 8 hours after intraperitoneal injection of hydrocortisone (5 mg per 100 g animal weight). The content of GC, physicochemical parameters (Tm, delta T, etc.) and DNA renaturation pattern did not show any changes. No changes were observed in the pattern of H3-thymidine incorporation into rat liver DNA: after hydrocortisone injection the radioactivity was found to be equally distributed in all isolated sequences of DNA, differing in the degree of reiteration (specific radioactivities of these DNA, fractions are very similar). Thus, the molecular population of DNA in liver cells remains unchanged, which suggests that the hormone-induced increase in the 5-methyl cytosine content is due to a change in the DNA methylation level. The methyation level of unique sequences (COt greater than 600), i. e. that of structural genes, does not undergo any essential changes. The reversible methylation of DNA regulated by hormones seems to be one of the mechanisms controlling gene activity.     

Cytophotometric comparisons of DNA levels in neuronal and glial cells of the cerebellum: A comparative study

Several cytochemical studies of the DNA content and ploidy status of neuronal cell nuclei in the central nervous system have reported the occurrence of hyperdiploid amounts of DNA in Purkinje cells and suggest the existence of some type of ‘extra’ DNA, the biological significance of which is, as yet, unknown. To explore this phenomenon further, the DNA content of glial and Purkinje cell nuclei was determined in several vertebrate species, using the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole (DAPI) to stain isolated cerebellar nuclei for analysis with a single parameter flow cytometer. The Feulgen reaction for DNA was used to stain liver and cerebellar tissue imprints for the measurement of individual nuclei with a Vickers M86 integrating microdensitometer. In both types of analyses, chicken erythrocyte nuclei served as an internal reference standard of 2.5 pg DNA per cell. The mean DNA content of Purkinje cells and glial or granule cells was essentially the same as that found for diploid (2C) non-neuronal cells, such as hepatocytes, in rainbow trout, Amazon molly fish, salamander (Plethodon), mouse, rat, rabbit, cat, dog, monkey and human. Although Purkinje cell nuclei with 4C DNA levels were found in all of these species, except salamander and rabbit, the frequency of such cells was low (1–7%) and varied with the species. There was a low incidence of Purkinje cell nuclei with interclass DNA amounts in all species examined. Our data show that most neuronal cell nuclei in the cerebellum contain 2C levels of DNA.     

Superficial urothelial (umbrella) cells. A potential cause of abnormal DNA ploidy results in urine specimens

OBJECTIVE: To determine the DNA ploidy distribution in urothelial superficial (umbrella) cells and to assess the value of the image analysis operator's experience. STUDY DESIGN: DNA ploidy was assessed in 12 cytologically negative bladder washes stained with Feulgen stain. All 12 cases were evaluated independently by three operators with different levels of cytopathology experience and different goals. Operator 1 (experienced) selected only nuclei of urothelial cells, avoiding nuclei of superficial cells; operator 2 (experienced) selected only nuclei of superficial cells; operator 3 (inexperienced) selected the largest and most-atypical-looking nuclei. Each operator measured a total of 100 nuclei per case. RESULTS: Operator 1 found all cases to be diploid (97% of nuclei on average). Operators 2 and 3 showed a wide range of results. Almost half the nuclei (47%) analyzed by operator 2 were in the diploid region, a third (35%) were in the tetraploid region, and the remaining (18%) ones had a DNA index (DI) in the range of 1.2-1.8 or > 2.5. Operator 3 obtained the most abnormal results. Only 9% of the nuclei were diploid, while 37% were in the tetraploid region, 18% were in the hyperploid region, and 35% had a DI in the range of 1.2-1.8. Differences among results obtained by each operator were statistically significant. CONCLUSION: The nuclei of superficial (umbrella) cells often have abnormal DNA content, which may cause abnormal DNA ploidy results in cytomorphologically normal bladder washes. Consequently, the nuclei of superficial cells should be avoided in the evaluation of urine samples. DNA analysis of urine specimens requires selection of nuclei only of deep urothelial cells by an experienced operator.     

DNA ploidy and S-phase fraction in carcinoma of the gallbladder related to histopathology, number of gallstones and survival.

Gallstones are a risk factor for the development of gallbladder cancer. We studied DNA ploidy and cell cycle composition by flow cytometry in archival specimens from 52 gall bladder carcinomas in relation to histopathological grade, tumour stage, gallstone number and survival. 69% of the gallbladder carcinomas showed aneuploidy. All tumours with single stones (N=11) were aneuploid while only 61% of tumours with multiple stones (N=41) were aneuploid (p=0.002). DNA aneuploidy was related to increase in T-category (p=0.01), grade (p=0.02), and nuclear pleomorphism (p=0.0005). The distribution of DNA ploidy shifted from tetraploid in low stage towards triploid positions in high stage tumours (p=0.02) combined with higher S-phase values in triploid tumours (p=0.05). S-phase fraction increased during development from normal tissue to dysplasia, cancer in situ and cancer in diploid cases (p=0.0002), and further at the change from diploid to aneuploid (p=0.004). At a median cancer specific survival time of four months patients with diploid tumours had a better survival than those with aneuploid tumours (p=0.02). In multivariate analysis of the tumour characteristic, only T-category and tumour grade were independent prognostic factors.The shift from diploid to aneuploid and the further shift of ploidy within aneuploid tumours are in agreement with the concept of a clonal development of gallbladder cancer. These changes are combined with a stepwise increase in the fraction of S-phase cells. Low frequency of symptoms in single stone patients may be the reason for detection of malignancy at a late stage of tumour development.     

Quantitative fluorescence imaging approach for the study of polyploidization in hepatocytes.

We applied automatic quantitative fluorescence imaging of nuclear DNA to rat liver cells obtained from animals at various times after birth up to 3 months of age. We show that, in conditions best preserving the native cellular structures, DNA content measurements, performed on whole single cells in situ after Hoechst staining, were precise and accurate. Cells in the various ploidy and nuclearity classes could thus be identified correctly and their percentages were estimated on a total of 300 cells or more. DNA synthesis was shown to occur asynchronously in all ploidy and nuclearity classes around weaning time. Observation of the labeling patterns, after in vivo BrdU pulse and short-term culture (chase), showed that the cell cycle was shorter in diploid cells compared with cells undergoing polyploidization. These results show that the approach of fluorescence imaging is well suited to investigations on polyploidization mechanisms.     

The reserve proteins in the cells of mature cotyledons ofLupinus albus var.Lucky

Summary The nuclear DNA content of cotyledonary cells of two lupin seeds (L₁ and L₂) with markedly different total protein content, were investigated by scanning cytophotometry. Both seeds had polyloid nuclei with DNA levels varying between 8 C and 64 C, the majority being either 16 C or 32 C. The highest DNA levels were found in the abaxial and central cotyledonary zones of both seeds; seed L₂ had a higher ploidy level than L₁. It is shown that the volume of condensed chromatin (chromocenters) increased proportionally with the DNA content of the nucleus. A comparison was made between the distribution of protein, previously determined byLe Gal andRey (1986) and the DNA throughout the cotyledon. The L₂ seed, which has the highest total protein and the highest protein content per cell, also exhibited the greatest DNA content per cell. For both seeds, the r-value for association of DNA and protein content per cell was highly significant (0.98).     

K-ras2 activation and genome instability increase proliferation and size of FAP adenomas.

The possible role of K-ras2 mutations and aneuploidy toward increase of proliferation and adenoma size in Familial Adenomatous Polyposis (FAP) adenomas is not known. The present study addresses these issues by investigating 147 colorectal adenomas obtained from four FAP patients. The majority of adenomas had size lower than or equal to 10 mm (86%), low grade dysplasia (63%), and were preferentially located in the right colon (60%). Normal mucosa samples were obtained from 19 healthy donors. Three synchronous adenocarcinomas were also investigated. K-ras2 mutation spectrum was analysed by PCR and Sequence Specific Oligonucleotide (SSO) hybridization, while flow cytometry (FCM) was used for evaluating degree of DNA ploidy and S-phase fraction. Overall, incidences of K-ras2 mutations, DNA aneuploidy and high S-phase values (>7.2%) were 6.6%, 5.4% and 10.5%, respectively. In particular, among the adenomas with size lower than 5 mm, K-ras2 mutation and DNA aneuploidy frequencies were only slightly above 1%. Statistically significant correlations were found between K-ras2 and size, DNA ploidy and size and K-ras2 and S-phase (p < 0.001). In particular, among the wild type K-ras2 adenomas, high S-phase values were detected in 8% of the cases versus 57% among the K-ras2 mutated adenomas (p = 0.0005). The present series of FAP adenomas indicates that K-ras2 activation and gross genomic changes play a role toward a proliferative gain and tumour growth in size.     

Can chromatin texture predict structural karyotypic changes in diploid cells from thyroid cold nodules?

To assess the effect of a single chromosomal translocation on the nuclear phenotype of human cells, seven diploid adenomas and five diploid carcinomas of the thyroid gland were studied using quantitative nuclear morphometry. Four adenomas and three carcinomas were cytogenetically normal, whereas three adenomas and two carcinomas had a unique chromosomal translocation. A densitometric parameter discriminated adenomas from carcinomas (skewness of the optical density histogram, SODH), and tumours with and without chromosomal translocation (standard deviation of the optical density, SDODH). These results demonstrate that single chromosomal structural rearrangements produce quantifiable alterations of nuclear organisation, but that other nuclear features which do not express an aneuploid DNA content or an abnormal karyotype differentially characterise benign and malignant conditions.     

Ploidy effects in isogenic populations of alfalfa : I. Ribulose-1,5-bisphosphate carboxylase, soluble protein, chlorophyll, and DNA in leaves

The influence of polyploidization on ribulose-1,5-bisphosphate carboxylase (RuBPCase), buffer-soluble protein (BSP), chlorophyll (Chl), and DNA was examined in fully expanded leaves of isogenic diploid-tetraploid (DDC 2X-4X) and tetraploid-octoploid (IC 4X-8X) sets of alfalfa (Medicago sativa L.). The concentration of RuBPCase in leaf extracts was determined by rocket immunoelectrophoresis. Activities of RuBPCase, expressed per milligram protein or per milligram Chl, and leaf tissue concentrations of RuBPCase, BSP, Chl, and DNA were similar between ploidy levels of the DDC 2X-4X set. Tetraploids and octoploids were similar in RuBPCase activities, expressed per milligram protein or per milligram Chl, and in leaf tissue concentrations of RuBPCase and DNA. Octoploids were significantly lower than tetraploids in concentrations of Chl and BSP.

When compared on a per leaf basis, tetraploids were 80% higher in BSP and essentially double comparable diploids in fresh weight, RuBPCase, Chl, and DNA. The observation that leaves of the DDC tetraploid population contain twice as much DNA as comparable diploids suggests that leaves of both ploidy levels contain similar numbers of cells. Leaves of the octoploid population were 33% to 80% higher than corresponding tetraploids in BSP, fresh weight, RuBPCase, Chl, and DNA. Ratios of RuBPCase to DNA and Chl to DNA were similar across ploidy levels of both isogenic sets suggesting that cellular content of Chl and RuBPCase increases proportionately with the amount of DNA per cell.

     

Human hepatocyte polyploidization kinetics in the course of life cycle

The processes of polyploidization in normal human liver parenchyma from 155 individuals aged between 1 day and 92 years were investigated by Feulgen-DNA cytophotometry. It was shown that polyploid hepatocytes appear in individuals from 1 to 5 years old. Up to the age of 50 years the accumulation rate of binucleate and polyploid cells is very slow, but subsequently hepatocyte polyploidization is intensified, and in patients aged 86–92 years the relative number of cells with polyploid nuclei is about 27%. Only a few hepatocytes in the normal human liver reach 16C and 8C×2 ploidy levels for mononucleate and binucleate cells respectively. Using a mathematical modeling method, it was shown that during postnatal liver growth the polyploidization process in human liver is similar to that in the rat, and that polyploid cells are formed mainly from binucleate cells. As in rats, prior to an increase in ploidy level, diploid human hepatocytes can pass several times through the usual mitotic cycles maintaining their initial ploidy level. After birth, only one in ten hepatocytes starting DNA synthesis enters the polyploidization process. At maturity about 60% of 2C-hepatocytes starting DNA synthesis divide by conventional mitosis, the rest dividing by acytokinetic mitosis leading to the formation of binucleate cells. During ageing the probability of hepatocyte polyploidization increases and in this period there are two polyploid or binucleate cells for every diploid dividing by conventional mitosis.