Microspectrophotometry of cell nuclei stained with the Feulgen reaction. IV. Formation of tetraploid nuclei in rat liver cells during postnatal growth

1. DNA contents of the individual parenchymal nuclei of rat livers during postnatal growth were estimated by microspectrophotometric apparatus, and different ploidy classes of nuclei were classified by their DNA contents. With the same material the total number of parenchymal nuclei in the liver was counted microscopically. 2. If the DNA content of nuclei encountered most frequently in several tissues represents the diploid class, the ploidy classes of the rat liver cell nuclei correspond to di-, tri-, tetra-, and octoploid, with the di- and tetraploid ones predominating considerably. 3. In suckling rats (below 25 gm. of body weight) the liver parenchyma is composed almost exclusively of cells with diploid nuclei, whereas in young rats (above 80 gm.), of tetraploid nuclei. In the growth stage between 25 and 80 gm., there is a remarkable replacement of the diploid nuclei by the tetraploid ones. However, in the liver of adult rats weighing more than 150 gm., any increase or decrease in the frequency of diploid and tetraploid nuclei is hardly observable. In such rats, the nuclear population of the liver parenchyma seems to reach a cell-ecological equilibrium which is considered to be a stable one. 4. It is shown that such nuclear populations and the total number of nuclei in a liver are controlled by the growth state, and not by the age. 5. The decrease in the total number of diploid nuclei and the increase in tetraploid nuclei in the growing livers of rats weighing from 40 up to 130 gm. can both be explained by the hypothesis that the tetraploid nuclei originate from the interphase diploid nuclei without involving mitosis. This hypothesis implies that mitosis is confined to the reproduction of diploid cells alone. 6. It is suggested that, in general, the synthesis of DNA does not necessarily result in the formation of visible mitotic chromosomes. 7. Mitotic time and generation time of diploid nuclei and the percentage of the tetraploidization from diploid nuclei are calculated and discussed.     

MICROSPECTROPHOTOMETRY OF CELL NUCLEI STAINED WITH THE FEULGEN REACTION : IV. FORMATION OF TETRAPLOID NUCLEI IN RAT LIVER CELLS DURING POSTNATAL GROWTH

1. DNA contents of the individual parenchymal nuclei of rat livers during postnatal growth were estimated by microspectrophotometric apparatus, and different ploidy classes of nuclei were classified by their DNA contents. With the same material the total number of parenchymal nuclei in the liver was counted microscopically. 2. If the DNA content of nuclei encountered most frequently in several tissues represents the diploid class, the ploidy classes of the rat liver cell nuclei correspond to di-, tri-, tetra-, and octoploid, with the di- and tetraploid ones predominating considerably. 3. In suckling rats (below 25 gm. of body weight) the liver parenchyma is composed almost exclusively of cells with diploid nuclei, whereas in young rats (above 80 gm.), of tetraploid nuclei. In the growth stage between 25 and 80 gm., there is a remarkable replacement of the diploid nuclei by the tetraploid ones. However, in the liver of adult rats weighing more than 150 gm., any increase or decrease in the frequency of diploid and tetraploid nuclei is hardly observable. In such rats, the nuclear population of the liver parenchyma seems to reach a cell-ecological equilibrium which is considered to be a stable one. 4. It is shown that such nuclear populations and the total number of nuclei in a liver are controlled by the growth state, and not by the age. 5. The decrease in the total number of diploid nuclei and the increase in tetraploid nuclei in the growing livers of rats weighing from 40 up to 130 gm. can both be explained by the hypothesis that the tetraploid nuclei originate from the interphase diploid nuclei without involving mitosis. This hypothesis implies that mitosis is confined to the reproduction of diploid cells alone. 6. It is suggested that, in general, the synthesis of DNA does not necessarily result in the formation of visible mitotic chromosomes. 7. Mitotic time and generation time of diploid nuclei and the percentage of the tetraploidization from diploid nuclei are calculated and discussed.     

Binding of the chemical carcinogen N-hydroxy-acetyl-aminofluorene to ploidy classes of rat liver nuclei as separated by velocity sedimentation at unit gravity

A large sedimentation device was developed that allows separation of 5 × 10⁸ rat liver nuclei by velocity sedimentation at unit gravity. Using the apparatus isolated rat liver nuclei were separated into classes of diploid stromal (Von Kuppfer, sinusoidal lining) nuclei, diploid parenchymal nuclei and tetraploid parenchymal nuclei respectively. DNA content and volume of the nuclei were measured. Diploid nuclei were 100% pure; tetraploid nuclei 98%.The in vivo binding of the liver carcinogen [³H]-N-hydroxy-AAF to these classes of nuclei was determined (total binding to protein, DNA and RNA). Binding and the subsequent removal of the fluorene derivatives was registered as a function of time. At all stages diploid stromal nuclei bound 2.6–5 times less carcinogen than did diploid parenchymal nuclei. Tetraploid parenchymal nuclei bound more than twice (2.3–3.95) the amount, that was present in their diploid counterpart. This effect became more pronounced 11 days after application of N-hydroxy-N-acetyl-2-aminofluorene.DNA was enzymatically purified from pooled classes of the various nuclear types. For purified DNA also it was found that DNA derived from diploid stromal nuclei bound 2.6–2.8 times less carcinogen than did DNA derived from diploid parenchymal nuclei.     

The nuclear dry weight of liver cells from patients with virus hepatitis and from controls.

Interferometric investigations were performed at liver cell nuclei isolated in 70 per cent glycerol. In 11 patients with virus hepatitis and seven patients without liver disease the nuclear dry weight of liver cells obtained by liver biopsy was determined by interferometry. The average nuclear dry weight of diploid liver cells from controls was 39.9 pg while an average value of 45.4 pg was found for patients with hepatitis. The corresponding nuclear volumes were 241 and 274mu3 respectively. The dry mass and volume of tetraploid nuclei was twice as big as that of diploid nuclei in both materials. The nuclear water content was neither significantly different between diploid and tetraploid nuclei nor significantly different between nuclei from controls and patients with hepatitis.     

The variation with age of nuclear phosphoproteins released during micrococcal-nuclease digestion and nucleosomal phosphoproteins in three cell types from rat liver.

The nucleosomal non-histone phosphoproteins, and phosphoproteins released during the digestion of nuclei by micrococcal nuclease, were studied in three rat liver nuclear populations, namely diploid stromal, diploid parenchymal, and tetraploid parenchymal nuclei, which were separated by zonal centrifugation, in 3-week-old rats in which the parenchymal cells contain diploid nuclei and in 2- and 4-month-old rats with increasing proportions of parenchymal tetraploid nuclei. Qualitative and quantitative differences in nucleosomal phosphoprotein band patterns were found among different types of nuclei and ages. More phosphoprotein bands were found in nucleosomes derived from parenchymal than stromal nuclei. The number of phosphoproteins released during micrococcal-nuclease digestion increased with age for parenchymal nuclei. The significance of these results, considered in conjunction with the increase of DNA repeat length and decrease of nuclease accessibility with age, is discussed.     

Cell populations of transplantable murine tumors of various degrees of histogenesis during their proliferation in the anterior chamber

Six transplantable murine tumors of different histogenesis were investigated after transplantation to subcutaneous connective tissue (SCT) and the eye anterior chamber (EAC). Cell morphology was studied using light microscopy. DNA contents in the nuclei of tumor cells were investigated with flow cytometry technique. LDH isoenzymes were studied using electrophoresis in polyacrylamide gel. In the case of tumors with near diploid modal class, a redistribution of LDH isoenzyme activity and an increase in morphological differentiation level were obtained. In the case of tumors with modal class differing from the diploid one, a morphological structure changes were revealed, but there were no differences in LDH isoenzyme activity. The data obtained show that the capability of increasing morphological and biochemical differentiation level after cultivation in the EAC of murine transplantable tumors remains even on the late stages of progression in tumors of different histogenesis with near diploid value of modal class.     

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.     

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.     

Biochemical,morphological and flow cytometric evaluation of the effects of hexachlorobenzene on rat liver

This study investigated the ability of HCB (0.1% in the diet for 15 days) to cause early changes in the cellular ploidy of rat liver. Treatment caused marked hepatomegaly, increase of microsomal proteins and cytochrome P-450 content and reduction of hepatocyte microviscosity. Microscopic examination showed that the hepatocytes were enlarged, with hyaline cytoplasm and vacuoles. The size distribution of the isolated hepatocytes showed a larger percentage of bigger cells. Flow-cytometric DNA/protein analysis was performed on whole (fixed) cells and on nuclei. From the combined results of both analyses it was possible to exclude significant changes in the percentages of diploid, mononucleated tetraploid, binucleated tetraploid and octoploid hepatocytes. The DNA and protein content of each subpopulation remained unchanged. Our results suggest that HCB does not cause early diploidization of liver cells and that hepatomegaly and cytochrome P-450 induction seem not to be correlated with effects on total DNA and total protein contents.Abbreviations HCB hexachlorobenzene - PI propidium iodide - FITC fluorescein isothiocyanate - DN diploid nuclei - SN 2N-4N nuclei in S-phase - TN tetraploid nuclei - DC diploid cells - SDC 2N-4N diploid cells in S-phase - TC tetraploid cells - STC 4N-8N tetraploid cells in S-phase - OC octoploid cells - MDC mononucleated diploid cells - SMDC mononucleated diploid cells in S-phase - BOC binucleated octoploid cells - SBTC binucleated tetraploid cells in S-phase - BTC binucleated tetraploid cells - MTC mononucleated tetraploid cells.     

Cytophotomeric DNA-analysis of aspirated cells from prostatic carcinoma.

In the present study material obtained from prostatic lesions by transrectal aspiration biopsy was subjected to a comparative morphologic and cytophotometric DNA analysis. Based on the morphologic pattern, the clinical material was divided into benign lesions (prostatic hyperplasia), suspected prostatic malignancy and highly, moderately and poorly differentiated prostatic carcinoma. The cytochemical analysis, based on quantitative cytophotometric measurements of Feulgen stained nuclei, showed that cell nuclei from benign lesions (prostatic hyperplasia) exhibited the normal diploid amount of DNA. Contrary to this, cell populations from prostatic malignancies, were characterized by various degrees of heteroploidy, i.e. the existence of cells with nuclear DNA quantities increased above the normal diploid level. A general correlation between degree of heteroploidy (frequency of heteroploid cells) and degree of clinical malignancy seemed to exist; high grade malignant prostatic carcinoma (poorly differentiated) exhibited a pronounced degree of heteroploidy with more or less distinct aneuploid stemlines, whereas low-grade prostatic carcinomas (highly differentiated) were more similar to benign cell populations, in showing a large proportion of cells with a diploid DNA quantity suggesting the existence of diploid or near diploid stemlines. Cases morphologically classified as moderately differentiated prostatic carcinoma, which previously have been shown to exhibit individual variations in degree of clinical malignancy, also showed large individual variations in degree of hetyroploidy. Approximately half of these cases had cytochemical DNA characteristics similar to that of highly differentiated prostatic carcinoma in showing a modal DNA value in the diploid region, while the other half showed cytochemical characteristics similar to that of poorly differentiated prostatic carcinoma, i.e. aneuploid DNA distributions. However, no morphologiifferentiated prostatic carcinoma. In conclusion it can be stated that the present investigation suggests the possibility that quantitative cytochemical DNA analysis may be used in combination with, and offer additional information to, morphologic analysis in the malignancy grading of prostatic carcinoma. A future clinical follow-up, now in progress, will hopefully give a more definite answer to that idea.     

Ploidy level and alpha-fetoprotein production in spontaneous murine hematomas in relation to the degree of histological differentiation

Comparative morphological, cytospectrophotometric and immunochemical studies were carried out on 26 hepatocarcinomas of various malignancy excised from 21 F1 (CBA X C57B16) mice. It has been shown that in morphologically similar hepatomas the ploidy level and alpha-fetoprotein production intensity may vary markedly. Highly differentiated carcinomas usually of smaller sizes and homogeneous morphology have the modal class nuclei with tetraploid (6 cases) or octaploid (3 cases) DNA content. In moderately- and low-differentiated carcinomas the diploid class of nuclei significantly increased (in 4 out of 6 tumours). Large tumours morphologically heterogeneous according to the differentiation level had polymodal DNA distributions with predominating tetraploid nuclei (II cases).     

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.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Summary Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

Early nuclear cytochemical changes in regenerating mammalian liver

Various cytochemical parameters were studied cytophotometrically in parenchymal cell nuclei isolated from rat liver at different times following partial hepatectomy. The study was confined to the early period following operation, before DNA synthesis and before mitotic activity ensued. Binding of acridine orange was found to increase approx. 70% over normal controls at 3 h post-hepatectomy followed by a sharp decrease to 60% below controls by 12 h and a return to near control levels at 24 h. These changes were found in both diploid and tetraploid cell nuclei. The initial two-fold difference in AO binding observed between diploid and tetraploid nuclei in normal liver persisted at 3 h but decreased markedly from 6 to 24 h after operation. Chromatin thermal stability, determined by acridine orange microfluorimetry, showed a rapid decrease at 3 h in both diploid and tetraploid cell nuclei followed by an increase above control levels at 6 h which persisted up to 24 h after partial hepatectomy. Parallel measurements of Feulgen dye binding showed no change in the relative DNA content of diploid and tetraploid cells throughout the experimental period. Ratios of specific picric acid and alkaline bromphenol blue dye binding by histone arginine and lysine side chains were found to rise significantly over normal controls at 3 h post-hepatectomy and return to normal levels by 6 h.     

Cytogenetics, flow cytometry, cytophotometry and morphometry of 22 cases of primary breast carcinoma. A comparative study.

Cytogenetic, flow cytometric, cytophotometric and morphometric analyses were performed on 22 previously untreated, primary solid breast carcinomas. Although the cell nuclei as the primary object of these studies were the same in all the tumors, distinct features were evaluated in each case to determine to what degree the results obtained by these techniques are comparable. From the cytogenetic viewpoint, six tumors had a modal number in the diploid range, seven were in the triploid range, and two in the tetraploid range; seven tumors had no modal number. These data correlate with the flow cytometry and cytophotometry results obtained, with DNA values slightly higher than their respective chromosomal modes. However, no correspondence between chromosomal modes and mean nuclear area was found. Chromosomal markers have been identified that particularly affect chromosomes 1 (p11, q21-qter), 11 and 16, although no common markers existed in all cases. Cytogenetics is the most sensitive technique, but the low yield (22 out of 140 tumors assayed) considerably restricts its value in any prospective breast cancer study.     

Flow cytometric analysis of isolated rat liver nuclei during growth

The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytometry using both scatter and fluorescent parameters to distinguish DNA diploid and DNA tetraploid populations and to discriminate between parenchymal and non-parenchymal compartments. The precise origin of each class of nuclei was assessed in whole liver homogenate using purified hepatocytes, obtained by liver perfusion followed by separation on Percoll gradient, and identifying the peaks corresponding to parenchymal nuclei. The results indicate that preparative procedures involving homogenization of the rat liver tissue caused loss of the DNA octaploid population. Data on the relative proportion of the different DNA ploidy elements during rat liver development, which are in good agreement with those observed by cell analysis by means of microspectrophotometry, indicate the usefulness of flow cytometry as a choice method for the analysis of ploidy distribution.     

Flow cytometric DNA and cytogenetic studies in human tumors: a comparison and discussion of the differences in modal values obtained by the two methods

The numerical chromosome values in 53 human tumors were determined and compared with the modal DNA values as measured by flow cytometry. In tumors with chromosome counts in the diploid and tetraploid range, the modal DNA values were found to correspond to the modal values based on the chromosome counts. In tumors with chromosome counts in the triploid range, however, the modal DNA values were about 15% higher than expected. In order to explain this difference, the ratio between large and small chromosomes in the karyotyped metaphases was assessed. In addition, the DNA content of individual chromosomes, including markers and minutes, was calculated as a reflection of the DNA content of the whole cell. The ratio of large to small chromosomes did not deviate from the normal ratio found in cells with diploid, triploid, and tetraploid chromosome counts. Neither difficulties in karyotyping nor short-comings in the flow cytometric methodology could be used to explain the discrepancy between the expected and empirical modal DNA values. Some of the chromosomes in triploid tumors may, therefore, contain an increased amount of DNA.     

Peculiarities of liver regeneration in Chinese hamster <Emphasis Type="Italic">Cricetulus griseus</Emphasis>

With the aid of cytofluorimetry and interference microscopy, the ploidy level and the hepatocyte ploidy class distribution were studied and the dry mass of hepatocytes was measured in hepatocytes in liver of Chinese hamsters Cricetulus griseus and of Balb/c mice before and one month after partial hepatectomy. The mean ploidy level in hepatocytes of the Chinese hamster normal liver amounted to 2.35 ± 0.03 c. The modal class was mononuclear hepatocytes with diploid nuclei (82.4 ± 1.3%). The mean dry mass of hepatocytes amounted to 605.2 ± 4.8 pg. In the process of liver regeneration in the Chinese hamsters, the ratio of ploidy classes and the hepatocyte dry mass did not change. After a similar liver resection in the mice, a significant polyploidization of liver parenchyma occurred. The mean ploidy level in hepatocytes rose by 32%. Instead of 4cx2-hepatocytes, the modal class became mononuclear octaploid cells the relative portion of which increased, on average, by five times. The portion of binuclear hepatocytes with octaploid nuclei in mouse liver rose by more than five times. Thus, in the Chinese hamsters Cricetulus griseus, unlike mice, regeneration of liver occurred exclusively at the expense of proliferation of hepatocytes.