DNA stainability in aneuploid breast tumors: comparison of four DNA fluorochromes differing in binding properties.

The aim of this study was to evaluate whether or not the differences in chromatin structure between diploid stromal cells or lymphocytes, which are often used as DNA ploidy standard, and aneuploid breast tumor cells can significantly affect the estimates of the DNA index of these tumors. To this end, the DNA content estimates of 34 aneuploid breast tumors, differing in size, degree of differentiation, and presence or absence of estrogen and progesterone receptors and metastases, were compared using four common DNA fluorochromes: DAPI, Hoechst 33342, propidium iodide, and acridine orange. These dyes differ in their mode of interaction with DNA (binding to minor groove or intercalation) and for each of them binding to DNA is restricted to a different degree by nuclear proteins. It was expected, therefore, that if differences in chromatin structure play a role in DNA content estimates, the DNA index of the measured tumors may vary depending on the dye. The cell nuclei were isolated from the tumors using a detergent-based procedure and stained with each of the dyes and the DNA index was estimated using peripheral blood lymphocytes as a DNA content standard. For each of the tumors, the DNA index estimates with all four dyes correlated very well. When the results obtained with individual dyes were compared in pairs, the correlation coefficients (r) of DNA indices were all above 0.96 (correlation at p less than 0.001). The best concordance was seen between specimens stained with Hoechst 33342 and DAPI (r = 0.99), and the least between those stained with Hoechst 33342 and propidium iodide (r = 0.96). The data indicate that DNA content analysis of unfixed nuclei, utilizing the above fluorochromes, is not significantly biased by differences in chromatin structure of the measured cells.     

Androgen receptor expression and DNA content of paraffin-embedded archival human prostate tumors

BACKGROUND: Androgen receptors (AR) are expressed in human prostate cells and immunohistochemistry has been used for qualitative analysis of AR expression in prostate tumor cells. Quantitative and multiparametric analysis of receptor expression could be of diagnostic and prognostic value in the management of patients on antiandrogen therapy. Multiparametric flow cytometric methods have been developed for analysis of hormone receptor expression and DNA content in nuclei isolated from formalin-fixed/paraffin-embedded human solid tumors. The present study was undertaken for analysis of AR expression and DNA content in archival human prostate tumors. METHODS: AR expression and DNA content were measured in nuclei isolated by enzyme digestion from thick sections cut from 51 paraffin-embedded human prostate tumors. AR expression in different subpopulations was studied by gated analysis. The relationship among AR activity, DNA content, and histopathological grade was analyzed. RESULTS: Distinct aneuploid populations were observed in 23% of tumors examined. AR activity was observed in all the specimens and the percentage of AR- positive nuclei in the 48 samples analyzed was <10% (n = 4), 11-50% (n = 39), and >51% (n = 5). Tumor subpopulations with aneuploid DNA content had higher AR expression (percent AR-positive cells and mean log fluorescence) than the diploid subpopulations. No strong correlation was seen between AR expression and histopathological grade of the tumors. CONCLUSIONS: Flow cytometric analysis of archival prostate tumor can be used for rapid determination of aneuploid DNA content and AR expression in subpopulations of nuclei isolated from formalin-fixed/paraffin-embedded prostate tumor blocks.     

DNA ploidy analysis of urinary tract epithelial tumors by laser scanning cytometry

OBJECTIVE: To evaluate a rapid and simple method for DNA content analysis of urinary tract epithelial tumors with laser scanning cytometry (LSC). STUDY DESIGN: The subjects were 25 patients (37 specimens) who underwent surgery for urinary tract epithelial tumors. Tissue specimens of such tumors were frozen immediately after tumor resection and stored at -80 degrees C until used. Touch preparations were made and fixed in ethanol at room temperature. The cell nucleus was stained with propidium iodide solution containing RNase, and DNA ploidy was analyzed by LSC. Nuclear debris and overlapping nuclei were gated out by special statistical filters. In LSC, a normal diploid reference peak was determined by observing lymphocytes morphologically on the computer display of the instrument and/or under the microscope. RESULTS: DNA ploidy could be evaluated in all tumor tissues. The time it took from preparing the tumor specimen to the last measurement was about 40 minutes at the shortest, and measurement of all the specimens was completed within one hour. The coefficient of variation was 2.8-7.8% (mean, 4.4%). All eight specimens (100%) at grade 1 (G1) were DNA diploid, but 20% and 85.7% of the G2 and G3 cells, respectively, were DNA aneuploid. In total, 15 of the 37 specimens were DNA aneuploid. All 17 pTa-pT1 specimens (100%) were DNA diploid, but 100% and 50% of the T2 and T3 tumors, respectively, were DNA aneuploid. CONCLUSION: One can now supplement a morphologic diagnosis with useful information using LSC of touch preparations of tumors obtained at surgery or of imprints of archived, frozen specimens. LSC provides excellent DNA histograms for surgical specimens and has great potential for clinical application in pathology.     

Two-color multiparametric method for flow cytometric DNA analysis of carcinomas using staining for cytokeratin and leukocyte-common antigen

Solid tumors contain heterogenous cell populations, resulting in flow cytometric (FCM) DNA quantitations of a mixture of tumor and host cells. Such mixed populations can result in dilution of the tumor cells by the host cells, in difficulty defining the diploid reference mean and in histogram peak overlap, precluding cell-cycle analysis. In this study, epithelial (tumor) cells and contaminating host cells in 100 consecutively accessioned human mammary and colorectal carcinomas were segregated in a multiparametric two-color FCM DNA analysis of intact, ethanol-fixed cells. These two carcinomas and bladder carcinomas contain a cytoskeleton of simple epithelium that is selectively stained with an FITC-labeled monoclonal antibody (MAb) to cytokeratin (CK: CAM 5.2-FITC). This MAb detects the CK 8, CK 18 and CK 19 consistently present in all layers of normal and neoplastic urothelium, colonic epithelium and mammary epithelium. Gating on CK in these tumors enables the nonstaining leukocytes, stromal fibroblasts and endothelial cells to be excluded from DNA analysis. A separate aliquot of each tumor evaluated was labeled with an MAb to leukocyte-common antigen (LCA-FITC) to serve as a patient-specific intrinsic diploid reference standard. Both the CK-labeled and LCA-labeled cells were then dual labeled for DNA with propidium iodide. This method (1) correctly identified the intrinsic diploid (LCA-positive) channel, allowing an accurate definition of normal cell DNA content for calculation of the DNA index; and (2) resulted in an increased sensitivity in the identification of both diploid and abnormal hyperdiploid tumor cell populations. It also (3) limited DNA cell cycle analysis to urothelial, colonic and mammary epithelial cells, the majority of which were neoplastic in carefully selected tumor samples. In addition, this method (4) clarified near-tetraploid populations that overlap the normal nonepithelial G2M region by diminishing the normal G2M peak and accentuating the aneuploid tetraploid G0G1 peak and (5) deconvoluted overlapping histograms composed of normal host and diploid-range or aneuploid tumor cells by gating on tissue-specific markers. This exclusion of host cells in both classes of tumors resulted in more accurate cell-cycle calculations in the former and allowed calculation of the S-phase fractions in the latter.     

A cytophotometric analysis of the structure of hypothalamic cell populations in the frog,Rana temporaria (L.), with special reference to seasonal changes in chromatin status

Summary We used cytophotometry after the Feulgen reaction and UV cytophotometry to measure the DNA content of quiescent cells of the hypothalamic preoptic region (HPR) of adult and juvenile frogs (Rana temporaria) that had been caught in their natural habitat in winter, spring and summer. The histone-to-DNA ratio in cell nuclei was cytophotometrically determined using a combined Feulgen, heparine and alcian-blue staining procedure. The vast majority of HPR cells studied had nuclei with a diploid DNA content. However, we observed great variability in the Feulgen-DNA content of the HPR cell population, which was not detected in the diploid standard (hepatocytes). This heterogeneity in the diploid sample of the HPR cell populations was always greater in prespawning frogs and may have been due to differences in the chromatin arrangement in nuclei. About 1% of cells had a DNA content either ranging between diploid and tetraploid levels (H2C cells) or at the tetraploid level (4C and 2C x 2 cells). The proportion of these cells was not affected by the age of the animals or the annual cycle, thus suggesting that there is no age-related increase in the mean DNA content in the frog HPR. The mean DNA contents of H2C and 4C cells were much higher than those in the standard (hepatocytes). This cannot be simply attributed to the presence of different amounts of nuclear proteins, but rather indicates that at least a certain proportion of the highest DNA contents may be due to a real extra-DNA synthesis.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

The variation with age of the structure of chromatin in three cell types from rat liver.

The organization of chromatin in three rat liver nuclear populations, namely diploid stromal, diploid parenchymal, and tetraploid parenchymal nuclei, which were separated by zonal centrifugation, was studied by digestion with micrococcal nuclease and pancreatic deoxyribonuclease in 3-week-old rats in which the parenchymal cells contain diploid nuclei and in 2-and 4-month-old rats with a high proportion of tetraploid nuclei. Digestion by micrococcal nuclease allowed the estimation of DNA-repeat length in chromatin. Parenchymal nuclei have shorter repeat length than stromal nuclei and DNA-repeat length increases with the age in all three nuclei populations. The kinetics of digestion by micrococcal nuclease showed that nuclei with shorter repeat length are more sensitive to micrococcal nuclease and that the sensitivity of chromatin decreases with age for all the types of nuclei in this study. The kinetics of digestion by pancreatic deoxyribonuclease showed that sensitivity of chromatin is related to the repeat length and that the sensitivity decreases with the ages.     

DNA content of Hurthle cell nodules in autoimmune thyroiditis

Hurthle cells are found in thyroid neoplasms and in reactive nodules in thyroiditis or goitrogenic processes. Cytometric studies have evaluated Hurthle cell neoplasms but not their reactive counterparts. DNA content of Hurthle cells in 22 cases of autoimmune thyroiditis was measured by flow cytometry and image content of Hurthle cells in 22 cases of autoimmune thyroiditis was measured by flow cytometry and image processing using nuclei extracted from paraffin-embedded tissue after microdissection of the Hurthle cell nodules. All 22 autoimmune thyroiditis Hurthle cell nodules were diploid, including 16 without associated neoplasms and six with associated malignant neoplasms (four papillary carcinomas, one follicular carcinoma and one follicular adenoma with papillary carcinoma). Concordance between flow cytometry and image processing was 100%. These findings indicate that the markedly atypical Hurthle cells in autoimmune thyroiditis are diploid by DNA quantitation. This suggests that atypia in Hurthle cells due to reactive or neoplastic processes may be differentiated by quantitative DNA analysis.     

Population of murine rhabdomyosarcoma MKh-53 clones under different proliferation conditions studied by flow cytometry

Clones of rhabdomyosarcoma cells were obtained due to implantation of the tumor cells in the eye anterior chamber, in subcutaneous connective tissue and in lungs of mice. The DNA contents in the clone cells were measured using flow cytometry. Diploid indices of clones were calculated from the ratio of the modal content of DNA in G1-cells to the DNA content in lymphocytes contaminating these clones. The diploid indices of various clones varied from 1.4 to 2.3. The mean diploid index calculated for clones that grew under given conditions of proliferation varied within 1.85-1.88. Only one of the 66 clones examined displayed a two-peak distribution of cell according to their DNA content in phase G1, which may suggest a karyotypic instability of the progeny of the tumor clonogenic cell. No correlation was revealed between the diploid indices of clones and the following parameters, such as: the portion of tumor cells being at different phases of cell cycle; the number of normal (stromal) cells contaminating the clones, coefficients of variation of DNA contents in tumor cell clones. A positive correlation was observed between the coefficients of variation of DNA contents in the normal (stromal) cells contaminating the clones and those of tumor clone cells being in phase G1. It is concluded that the variability of results of the flow cytometric measurement of DNA in G1-cells may reflect the variability of cells in respect not only to their DNA contents but also to their capacity of dye sorbtion.     

Cytofluorometric nuclear DNA content analysis of breast tissues after frozen section diagnosis

OBJECTIVE: To establish a suitable method for measurement of nuclear DNA content in breast tissues from frozen storage after frozen section diagnosis. STUDY DESIGN: For fundamental research, rat liver samples preserved in a deep freezer were used. Four protocols were used (1. fixation with 70% ethanol followed by naked nuclei preparation; 2. fixation with 10% neutral buffered formalin followed by naked nuclei preparation; 3. preparation for naked nuclei prior to fixation with 70% ethanol; and 4. preparation for naked nuclei prior to fixation with 70% neutral buffered formalin). For clinical research, 13 separate fresh frozen breast tissue samples were analyzed after frozen section diagnosis. One contained a malignant phyllodes tumor (MPT) consisting of 2 components, benign epithelial cells and malignant stromal cells; 3 were benign tumors containing fibroadenoma; and 9 cases were carcinomas, consisting of 5 scirrhous, 3 papillotubular and 1 mucinous. RESULTS: Protocols 1, 2 and 3 were not suitable methods for our purpose because remaining cytoplasm or cohesive nuclei were observed. In protocol 4 the cytoplasm was completely undetectable, and nuclei were suitably separated for nuclear DNA content measurement. Benign epithelial cell component nuclei presented a diploid pattern, and the malignant stromal cell component nuclei indicated a euploid pattern in MPT. All 3 cases of benign constituents in fibroadenoma showed a diploid pattern, as did the 3 carcinoma cases (1 mucinous, 1 scirrhous and 1 papillary). Four scirrhous and 2 papillary carcinomas showed an aneuploid pattern. CONCLUSION: Our findings show that it is possible to measure nuclear DNA content of human frozen storage tissues after frozen section diagnosis.     

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.     

Flow cytometric analysis of DNA in cells obtained from deparaffinized formalin-fixed lymphoid tissues

Flow cytometric analysis of DNA content was performed on nuclear suspensions prepared from fresh and from paraffin-embedded, formalin-fixed lymphoid tissues. We confirmed previous reports that it is possible to obtain nuclear suspensions from deparaffinized, formalin-fixed tissues, suitable for DNA analysis by flow cytometry. We observed a tendency for a larger coefficient of variation (CV) of the DNA measurements in the fixed tissues than in the unfixed material causing abnormalities in 2 of 19 lymphomas to become undetectable. Furthermore, samples from different paraffin blocks of a single tumor with an extra G1 (hyperdiploid) peak showed marked differences in the CV of the hyperdiploid peak while the CV of the diploid peak was similar in all samples. In both benign and malignant lymphoid tissues, the S-phase fraction was higher in paraffin-embedded tissues than in unfixed cells. This difference could be attributed to 4', 6'-diamidino-2-phenylindole dihydrochloride (DAPI), a DNA-binding dye commonly used in this technique. Nevertheless, intermediate and high grade lymphomas from paraffin-embedded tissues generally showed a greater S-fraction than low grade lymphomas, a similar observation as with unfixed tissues. Therefore, DNA content analysis of nuclei extracted from paraffin sections may be inadequate to resolve slight aneuploidy, but the measurement of S-fraction size may remain diagnostically or prognostically valuable. Large retrospective studies will be necessary to determine the clinical impact of this technique in the analysis of lymphomas.     

Determination of ploidy and steroid receptor status in breast cancer by laser scanning cytometry

BACKGROUND: Measurements on DNA content and steroid receptor status in breast cancer are of great clinical interest. Objective determination of estrogen and progesterone receptor expression should help to define the lowest levels of positivity still responding to adjuvant antihormonal therapy. For this purpose, a simple protocol for laser scanning cytometry is presented. METHODS: Analysis of 54 routine breast cancer samples was performed by laser scanning cytometry (LSC). To obtain single cell preparations from fresh tumor tissue, slides were prepared using the Cervisoft cytological device. Exact determination of tumor cell DNA content was done by referring to the CD45-positive tissue leukocyte fraction as the internal diploid reference cell population. Steroid receptor-expressing cells were detected by indirect immunolabeling. RESULTS: Indirect immunofluorescence allowed the best quantification of both the estrogen and progesterone receptor-expressing cell fractions by LSC. The number of receptor-expressing cells could be given in percentage. For comparison, the 10% cutoff value was used to determine receptor positivity. CONCLUSION: LSC enabled a simple, reliable, and inexpensive determination of DNA index and steroid receptor expression in breast cancer specimens by objective criteria.     

Cytophotometric analysis of the cell population composition of the the preoptic area of the hypothalamus in the common frog

DNA contents in squashed cells of the adult frog hypothalamic preoptic region (HPR) were measured using the Feulgen and UV cytophotometry techniques. The histone-DNA ratio in the cell nucleus was determined by means of a combined Feulgen-heparin-Alcian blue staining procedure. The nuclei of the vast majority of HPR cells have a diploid DNA content. However, in cells of this group the mean values of DNA amount and the distribution range were always higher than those in hepatocytes used as a diploid standard. Such a heterogeneity in DNA content in the diploid part of HPR cell population could apparently suggest some differences in the nuclear chromatin arrangement to be always higher in spring before the frog spawning, and it seems to be characteristic of this type of cells. About 1 per cent of cells with hyperdiploid surplus of DNA (H2c cells) as well as of tetraploid cells (4c and 2c X 2 cells) is found in HPR in frogs sacrificed both in winter and in summer. The quota of these cells has no reference either to the frog's age or to the annual cycle. The fact that the mean DNA values in H2c and 4c cells are much higher than in the standard cannot be explained by the presence of different amounts of nuclear proteins only. It is suggested that at least some part of the highest DNA values may be due to an actual extra DNA synthesis in a small constantly existing pool of HPR cell population.     

Exfoliative cytology of a lymphoepithelioma-like carcinoma in a cervical smear. A case report

BACKGROUND: Lymphoepithelioma-like carcinoma of the cervix (LELC) is cytologically identical to its counterparts at other sites, such as the nasopharynx. LELC can be suspected on a cervical cytologic smear. The differential diagnosis includes nonkeratinizing squamous cell carcinoma with prominent stromal inflammation, carcinoma with intense stromal eosinophilia, glassy cell carcinoma, malignant lymphoma (especially lymphoepitheloid-Lennerts lymphoma) and metastatic Schmincke-Regaud tumor. CASE: A 55-year-old female presented with an ulcerated endophytic tumor in the cervix. Exfoliative cytology showed uniform, large tumor cells, often associated with inflammatory cells, with round or oval nuclei and one or more prominent nucleoli. The cytoplasm was finely granular to flocculent, and the nuclei were uniformly vesicular. The chromatin was peripherally marginated. The cell borders were indistinct. There was no evidence of dyskeratotic or keratinized cells, koilocytes or glandlike formations. These findings were highly suspicious for LELC and were confirmed by biopsy. Flow cytometry showed DNA aneuploidy, with a DNA index of 1.08. In situ hybridization was negative for human papillomavirus 16 and 18. CONCLUSION: LELC of the uterine cervix has cytologic features that are sufficiently characteristic for a specific cytologic diagnosis. The diagnosis, nevertheless, has to be proven by histology.     

Nucleoprotein changes in plant tumor growth

Tumor cell transformation and growth were studied in a plant neoplasm, crown gall of bean, induced by Agrobacterium rubi. Ribose nucleic acid (RNA), deoxyribose nucleic acid (DNA), histone, and total protein were estimated by microphotometry of nuclei, nucleoli, and cytoplasm in stained tissue sections. Transformation of normal cells to tumor cells was accompanied by marked increases in ribonucleoprotein content of affected tissues, reaching a maximum 2 to 3 days after inoculation with virulent bacteria. Increased DNA levels were in part associated with increased mitotic frequency, but also with progressive accumulation of nuclei in the higher DNA classes, formed by repeated DNA doubling without intervening reduction by mitosis. Some normal nuclei of the higher DNA classes (with 2, 4, or 8 times the DNA content of diploid nuclei) were reduced to diploid levels by successive cell divisions without intervening DNA synthesis. The normal relation between DNA synthesis and mitosis was thus disrupted in tumor tissue. Nevertheless, clearly defined DNA classes, as found in homologous normal tissues, were maintained in the tumor at all times.     

Cytophotometric and electron microscopic studies of chronic human skin lymphomas. II. Research on lymph node involvement

Results of electron microscopic and cytophotometric studies of biopsy material from lymph nodes of patients with cutaneous lymphomas with low degree of malignancy are discussed, with special reference to early diagnostic criteria of the disease. Specific characteristics of tumor tissue involve the presence of atypical lymphocytes with marginal condensed chromatin extrusions of nuclear material, deep invaginations of the nuclear membrane, nuclear pockets, excess of the mean DNA content per nucleus above the diploid standard level, more than 5% of nuclei being in the hyperdiploid area. Electron microscopic and cytophotometric methods allowed to diagnose the tumor injury of lymph nodes, when the traditional histological techniques revealed no signs of malignancy.     

THE NUCLEAR DNA CONTENT OF LARGE VENTRAL SPINAL NEURONS

Abstract— A nuclear fraction has been obtained from large spinal neurons previously isolated from bovine ventral spinal cord in bulk suspensions. The fraction contained an average of 5.3 ± 0.9 pg DNA/nucleus, indicating a high incidence of diploid nuclei. This conclusion was confirmed by distribution analysis of DNA in propidium iodide-stained nuclei examined by flow microfluorometry. That technique showed that at least 90% of the nuclei from large spinal neurons are diploid. Mixed, mostly non-neuronal nuclei derived from many types of cells in the ventral spinal cord contained an average of 5.9 ± 0.6 pg DNA/nucleus, 19% of which possibly possess more than diploid amounts of DNA. The uniform DNA content in nuclei of large spinal neurons and most other types of cells in the ventral spinal cord contrasts sharply with a wide variation (av 26-fold) in the nuclear volumes of the same cells.     

Cytological studies on the organization of DNA in giant trophoblast nuclei of the mouse and the rat

Giant trophoblast nuclei of the mouse and the rat, known to contain hundreds, or even thousands, of times the haploid amount of DNA, have been studied by a number of cytological techniques. These nuclei appear in two morphological states:“reticulate,” in which large numbers of chromatin threads of uniform size intermingle throughout the nucleus, often radiating from clumps of heterochromatin adjacent to the nucleoli, and “homogeneous,” in which the chromatin is more evenly dispersed and individual threads are more difficult to distinguish. Intermediate morphologies are also observed. In neither case were structures resembling polytene chromosomes discernible. — Centromeric heterohromatin as revealed by the Giemsa BSG technique has been quantitatively analyzed in giant versus diploid trophoblast nuclei. Although the median number of chromocenters is slightly greater in giant than in diploid nuclei, the range is similar. In both cases, the chromocenter number is usually less than the diploid number of chromosome pairs, indicating the attraction between centromeres not only of homologous, but also of heterologous, chromosomes. By scanning microdensitometry, we have observed a constant ratio of chromocenter area: total nuclear area in giant cells. This ratio, which likely reflects the ratio of chromocenter volume: total nuclear volume, is in good agreement with that of satellite DNA: total DNA.     

Heterochromatin,repetitive DNS und Nukleolen in Leberzellen von Microtus agrestis

Zusammenfassung Die Zellstruktur von Leberzellen der Erdmaus, Microtus agrestis, wurde nach Giemsafärbung, Feulgenbehandlung, Behandlung mit Ribonuklease und nach Färbung des konstitutiven Heterochromatins untersucht. Das konstitutive Heterochromatin ist in Leberzellen nicht heteropyknotisch, das fakultative Heterochromatin ist im weiblichen Geschlecht als Sexchromatinkörperchen sichtbar. Bestimmungen des relativen DNS-Gehalts ergaben, daß die Zahl der Sexchromatinkörperchen der Ploidie der Zellkerne proportional ist. Die Nukleolen liegen in Hepatozyten oft randständig; in 59% der diploiden Zellkerne sind 2 Nukleolen enthalten. Nach Anfärbung der repetitiven DNS werden oft auch die Nukleolen gefärbt, nach Ribonukleasebehandlung tritt dieser Effekt nicht auf. Das konstitutive Heterochromatin wird in Form von 2 langen fädigen Strukturen sichtbar.

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Heterochromatin, repetitive DNA and nucleoli in liver cells of Microtus agrestis

Summary The nuclear structure of parenchymal liver cells of embryo and adult Microtus agrestis was studied in smear and section preparations after staining with Giemsa solution and treatment according to Feulgen, after treatment with ribonuclease and after specific staining of constitutive heterochromatin. In liver cell nuclei only the facultative heterochromatin is heteropycnotic, a sex chromatin body is observable in female but not in male animals. Constitutive heterochromatin is not heteropycnotic in liver cells. Measurements of the relative DNA content showed that nuclei with one sex chromatin body are diploid; tetraploid nuclei possess two and octoploid nuclei four sex chromatin bodies. Solely in the diploid cell nuclei of the intrahepatic gall ducts two large chromocenters are found. The nucleoli in hepatocytes often lie at the perimeter of the nucleus. 17% of the diploid nuclei contain one nucleolus, 59% two nucleoli, 23% three and 1% four. After staining of repetitive DNA, the nucleoli often become stained as well; after treatment with ribonuclease this effect does not occur. The constitutive heterochromatin becomes visible in form of two long, threadlike structures. After longer periods of dissociation the sex chromatin body ceases to be visible. Sex chromatin and constitutive heterochromatin are contiguous to the nucleoli.

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Mit dankenswerter Unterstützung durch das Bundesministerium für Bildung und Wissenschaft der Bundesrepublik Deutschland.     

Distribution of 5-methyldeoxycytidine in products of staphylococcal nuclease digestion of nuclei and purified DNA

We have compared the distribution of 5-methyldeoxycytidine (m5dC) between staphylococcal nuclease (SN) sensitive and resistant regions of human diploid fibroblast chromatin to the corresponding distribution in purified DNA. After SN digestion of fibroblast nuclei or purified DNA, nuclease-resistant products were separated from sensitive products by perchloric acid or ethanol precipitation; the radioactively labeled nucleosides were then fractionated by high-performance liquid chromatography and quantitated. Our results indicate that m5dC is preferentially associated with SN-resistant regions of both chromatin and purified DNA. The magnitudes of these preferences in fibroblast chromatin and DNA are similar; we find that the enrichment of m5dC content in SN-resistant fractions of nuclei and DNA relative to the corresponding sensitive fractions is approximately 2-3-fold. Therefore, highly methylated regions of DNA have an intrinsic resistance to digestion by SN that is of sufficient magnitude to explain the high degree of nuclease resistance of chromatin containing highly methylated DNA.