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Determination of DNA content in the nurse and follicle cells from wild type and mutant Drosophila melanogaster by DNA-Feulgen cytophotometry.

DNA replication patterns in the nurse and follicle cells of wild type and a female sterile mutant, fs(1)1304, of Drosophila melanogaster have been studied by DNA-Feulgen cytophotometry, using a cell dispersal technique that allowed the measurement of DNA amounts in individual nuclei from egg chambers of known developmental stages. DNA-Feulgen values associated with various ovarian nuclei from egg chambers at different stages of development were used to assess a base line DNA content for ovarian tissues and to estimate the extent of DNA replication in the nurse cells and follicle cells of growing and mature egg chambers. Our data show that both the nurse and follicle cells undergo multiple cycles of endonuclear DNA replication and that there may be selective amplification as well as underreplication by portions of the genome in these highly polyploid, ovarian cells. Alternative models are proposed to account for the DNA replication patterns observed. Comparisons of DNA-Feulgen levels in wild type ovarian nuclei with those found for the fs(1)1304 mutant and its heterozygote in the balanced stock fs/FM3, show that equivalent DNA levels are present in follicle cell nuclei from all three types of females. Nurse cell nuclei in the homozygous fs stock, however, fail to achieve the same high DNA levels observed in both fs/FM3 and wild type nurse cell nuclei. Although the nuclei of follicle cells in ovaries from fs/fs females appear morphologically like those surrounding egg chambers in wild type ovaries, nurse cell nuclei from mutant females show a more compacted organization of their chromatin than found for nurse cell nuclei from wild type ovaries at similar developmental stages. Our findings suggest that a major effect of the fs(1)1304 mutation may be on the coiling behavior of chromatin and the conformation of DNA-protein moieties in both nurse cell and follicle cell nuclei. These changes in chromatin structure apparently are manifest by perturbations in DNA replication patterns and normal gene function in these biosynthetically active cells.

Animals↗

HIDACSYS: computer programs for interactive scanning cytophotometry.

A description is given of a combination of three programs developed for computer-assisted stage scanning cytophotometry and cytofluorometry of isolated, close-lying, or touching objects in different types of microscopical preparations. The advantages and limitations of the individual programs are discussed, as well as the local specimen conditions determining the optimal application range of each of the programs. The applicability of these programs was investigated by determination of the integrated absorbance values of Feulgen or gallocyanin-chrome alum stained chicken erythrocytes, human leucocytes, and skin biopsy cells in imprint preparations, as well as of guinea pig peritoneal granulocytes which had been submitted to a simultaneous coupling azo dye incubation for alkaline phosphatase activity.

Alkaline Phosphatase↗

Dual wavelength scanning cytophotometry (Bicoscan).

A computer program has been developed for stage-scanning cytophotometry of double-stained microscopical specimens. The program permits the simultaneous measurement of absorbance values at two wavelengths in each measuring spot. To account for overlap in the absorbance spectra of the two stained endproducts, the program incorporates correction of the measured data to compensate for the contribution of each chromophore to the absorbance measured for the other. The program will compute the corrected local absorbance values at specified wavelengths for each chromophore at each measuring spot and integrate these values over the total object to give separate totals for each stain. It is also possible to have integrated the absorbance values of one chromophore for all those measuring spots where the local corrected absorbance value of the other chromophore exceeds a preset minimum value. When this other chromophore is a nuclear DNA stain, it is possible to obtain an approximate measure of the content of any compound in the nuclear area which can be stained with a chromophore having an absorbance spectrum different from the DNA stain. The validity of the program was investigated on model preparations consisting of two (differently) coloured films of which the absorbance values could be measured either individually or in combination, by partially overlaying one film on the other. The program's potential has been demonstrated by using the combination of either Naphthol Yellow S or dinitrofluorobenzene as protein stains with the Feulgen-pararosaniline(SO2) procedure for DNA in chicken erythrocytes and rat liver cells.

Animals↗

DNA-cytophotometry and immunocytochemistry in ovarian tumours of borderline malignancy and related peritoneal lesions.

A total of 34 surgical specimens, obtained from 13 patients with ovarian tumours of borderline malignancy (OTBM), were investigated by conventional histology, immunocytochemistry and DNA cytophotometry. The lesions were obtained by primary ovarian surgery or second-look procedures and altogether comprised 19 (single and bilateral) OTBM, 8 cases of endosalpingiosis, 4 in situ and 2 invasive peritoneal implants and 1 overt adenocarcinoma. The morphological findings were related to follow-up data, which showed neoplasms with clinically malignant behaviour in 2 patients. The histology of the extra-ovarian manifestations was not associated with their immunocytochemical properties or with their DNA content. There were no correlations between the evolution of disease and microscopical features but the clinical course appeared to be linked to the DNA content of the extra-ovarian lesions, which was of greater prognostic importance than DNA ploidy of the ovarian tumours. Recurrence-free survival was noted in all 5 patients with diploid or euploid extra-ovarian proliferations, while the 2 clinically malignant cases fell into the group of 3 patients with noneuploid or aneuploid specimens. DNA estimations may be a methodology which increases the prognostic value of second-look procedures in OTBM patients.

Adult↗

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.

Adult↗

[Morphology, DNA cytophotometry and prognosis in gastrointestinal neuroendocrine tumours (carcinoids). A clinico-pathological study of 95 patients].

A total of 123 manifestations (97 primary tumours and 26 metastases) of neuroendocrine tumours of the gastrointestinal tract observed in 95 patients was investigated for the prognostic value of clinical, histological and DNA cytophotometric parameters. Metastases almost exclusively occurred among ileal carcinoids, which also were responsible for all 14 cases of lethal outcome observed during the follow-up period of mean 42 months. Aneuploid DNA values could be determined significantly more frequently among ileal than in non-ileal carcinoids and showed - upon analysis of the total group of gastrointestinal neuroendocrine tumours - a significant correlation to lethal course of disease. In addition, among 18 cases with primary and secondary carcinoid manifestations available for DNA cytophotometry, an association between the DNA content of metastatic neuroendocrine tumours and prognosis came to light. When applied to the group of ileal neoplasms, however, the parameter DNA content did not allow a better prognostic assessment.

Adult↗

The problem of cell debris in pulse-cytophotometry: a probability theoretic model.

The problem of cell debris in pulse-cytophotometry is considered on the basis of a probability-theoretic model and the results are compared with the model of a pure exponential function for the portions of the DNA fragments in the channels of the flow cyclometer, which is generally used in practice. The model is based on the assumption that the possible decomposition of the DNA content of a single cell into some parts as a consequence of the necessary preparation of the cell material for measurement can be interpreted as a Poisson point process. Therefore the number of divisions of the DNA content of a cell is assumed as a Poisson random variable. It follows a well-defined distribution function of the DNA content in the common population of intact cells and fragments depending on the apriori distribution before pretreatment. This distribution determines a theoretical histogram which can be compared with the measurements. The results corroborate the assumption of a pure exponential course for the portions of DNA fragments only in the area of very small DNA contents. For greater DNA contents there are differences between the pure exponential course and the model described here depending on the a priori distribution and on the intensity of the cell disintegration process. As a byproduct, an estimation of the fraction of disintegrated cells is obtained with respect to the fraction of fragments. A corresponding computer program was applied to a number of experimental histograms. The results have shown that the theory describes the measured histograms satisfactory in most cases.

Cell Cycle↗

Characterization of cells of the normal aortic endothelium of adult rats and changes due to endotoxin shock. I. Communication: light microscopy, autoradiography, DNA cytophotometry, and enzyme histochemistry.

Aortic endothelium of normal and endotoxin treated rats was investigated in "Häutchen" preparations by light microscopy, autoradiography, DNA cytophotometry, and enzyme histochemistry. In the endothelium of control rats the following cell populations were found: 1. Endothelial cells (90% of all cells in endothelium), characterized by the alignment of the longitudinal axis of their nuclei, their light microscopic morphology, their DNA content resp. DNA synthesis, and by their enzymatic activity; 2. mononuclear cells (10% of all cells in endothelium), identified as mature blood monocytes, mononuclear phagocytes (macrophages), and as lymphocytes; 3. granulocytes (less than 1% of all cells in endothelium). The presence of mononuclear cells and granulocytes in the normal endothelium appears to be the expression of a physiological repair process in endothelium. After endotoxin application one can observe an endothelial damage and then a repair process. During the repair of endothelium the following processes occurred: An increase of DNA synthesis particularly of endothelial cells, an aggregation of platelets on the endothelial surface, an increase in number of granulocytes and at last an increase in number of activated mononuclear cells. Participation of mononuclear cells in the physiological as well as in the pathological repair process of endothelium was shown by the present investigation. The process of transformation of mononuclear cells into endothelial cells is discussed at the same time.

Animals↗

Bone-marrow-profileration patterns in acute myeloblastic leukaemia determined by pulse cytophotometry.

The proliferation patterns of normal and leukaemic bone-marrow were studied by measuring the D.N.A. content of large numbers of cells by pulse cytophotometry (P.C.P.). In nineteen normal bone-marrow samples an average of 66-3% of the bone-marrow cells were in the G1 phase (2n D.N.A.), 26-1% in the S phase (2n smaller than D.N.A. smaller than 4n), and 7-5% in G2+M phase (4n D.N.A.). The percentages of S-phase cells determined by autoradiography and P.C.P. correlated well, both in normal and in leukaemic bone-marrow. In 25 patients with untreated acute myeloblastic leukaemia (A.M.L.) lower percentages of cells were found in S and G2+M phases, indicating a smaller proliferating pool compared with normal bone-marrow. The likelihood of a complete remission being attained in A.M.L. with the first treatment course was correlated with the percentage of S-phase cells present before treatment. At remission in A.M.L. the proliferation pattern was restored to normal.

Autoradiography↗

Early responses to chemotherapy detected by pulse cytophotometry.

DNA/cell distributions were recorded by automated cytofluorometry (=pulse cytophotometry) in bone-marrow aspirates of leukaemia and lymphosarcoma patients subjected to chemotherapy. In most cases, early perturbations in DNA/cell histographs were observed, characteristically reflecting the known mode of action of the drugs. These changes in general preceded the clinical observation of drug response. In a series of 23 measurements in 19 patients, a positive correlation between early cytophotometric changes and clinical effects of chemotherapy was observed in 17 patients. Five patients were negative for both cytophotometric and clinical reactions and one patient was probably false-positive. The validity of the assay for early detection of drug resistance in acute leukaemia and related diseases is discussed.

Adult↗

[DNA measurement of malignant tumors by impulse cytophotometry. The principles and importance for assessing growth behavior and the degree of abnormality].

Monometric DNA impulse cytophotometry of ploidism state (diploid or aneuploid tumors with stemmline shift, polyploidism ) and proportion of DNA-synthesized cells (S-phase proportion) were determined on over 500, mostly malignant, tumors of different sites. The results were compared with histopathological findings (TNM stage and degree of differentiation). Large diploid carcinomas of the oral cavity have lower S-phase activity than aneuploid tumors. Carcinomas of the breast, stomach and ovary are frequently diploid. Aneuploid carcinomas of these regions generally have high synthesizing activity. Mucoid signet-ring carcinomas of the stomach have lower S-phase activity than non-mucoid carcinomas. In the colorectal region aneuploid carcinoma predominates, in the colon more than in the rectum. Tumor metastases from colon and rectum into the liver predominantly are from aneuploid primary tumors. For tumors of the breast, corpus uteri and ovary with high and moderate differentiation there is a direct correlation between histological grade of differentiation and the S-phase proportions. Aneuploid tumors vary from low to high synthesizing activity. Diploid meningioma and glioma have a low S-phase proportion, aneuploidism correlates with an increase in growth. Supplementation of histopathological diagnosis by determining ploidism and S-phase activity makes an important contribution in the assessment of the degree of malignancy, as well as for therapeutic purposes.

Aneuploidy↗

Correlations between ploidy and initiation probability determined by DNA cytophotometry in individual altered hepatic foci.

Single cell DNA cytophotometry of ATPase-deficient putative preneoplastic foci in rat liver, induced by a single dose of N-methyl-N-nitrosourea (25 mg/kg or 50 mg/kg) and subsequent phenobarbital feeding (0.05% in the diet), revealed three different types: the majority of foci consisted of an almost exclusive diploid cell population, others showed a preferential tetraploid pattern and a few large foci contained a mixture of di-, tetra- and octoploid hepatocytes. The results of this selective measurement of individual preneoplastic foci in Feulgen-stained 20-microns-thick cryostat sections indicate that not only diploid hepatocytes are a target for the transforming action of a carcinogen, but also tetraploid cells, and that both initiated cell types are capable of expanding as a homogeneous clone. However, clonal homogeneity appears to be lost when foci enlarge. A few preneoplastic foci, heterogeneous with respect to DNA content and endowed with an increased proliferative potential, may represent cell populations at high risk of further development into malignancy.

Adenosine Triphosphatases↗

Cytophotometry of liver cells from ethanol-fed rats: ethanol causes increased polyploidization and protein accumulation.

Cellular protein and DNA content were determined by Feulgen--Naphthol Yellow S cytophotometry on isolated liver cells from rats fed ethanol for 2--6 months. It was found that parenchymal liver cells after ethanol feeding contained 13% more protein than cells of the same ploidy class from controls; that the average parenchymal cell in ethanol-fed rats contained 16% more protein than in controls due to the increased occurrence of polyploid cells; that the protein accumulation develops over 2 months, does not progress thereafter and disappears after a few days of reduced ethanol intake; that the tendency to form polyploid cells is accentuated during liver regeneration. It is suggested that the effect of alcohol on microtubular function, which is regarded as the cause of the protein accumulation, may also bring about increased polyploidization.

Animals↗

A consideration on histopathologic variability of diffuse pleural mesothelioma based on DNA cytophotometry.

Nuclear DNA cytophotometry of the tumor cells was performed for an autopsy case of malignant diffuse pleural mesothelioma diagnosed histologically as a mixed type. The DNA distribution of these tumor cells confirmed bimodality, one being situated in the triploid and the other in the pentaploid DNA range, thus indicating two stemline cell populations. In the former, the majority were epithelial mesothelioma cells resembling the normal mesothelial cells. The latter were mesenchymal tumor cells with spindle and occasionally bizarre shaped nuclei. This cytophotometric data strongly suggests that the histologic variability in mesotheliomas results probably from the change in number and ploidy level of the stemline cell population accompanied by neoplastic development.

Adult↗

A study on the mesothelial cell kinetics in pleural effusions by DNA cytophotometry and autoradiography with tritiated thymidine.

On 34 samples of pleural fluid a cell kinetic study of mesothelial cells was carried out by Feulgen-DNA cytophotometry and the autoradiographic estimation of 3H-thymidine uptake. The generation time of mesothelial cells in malignant effusions was found to be shorter than in non-malignant ones, thought he cellular activity for both effusions was essentially the same. Furthermore, with the development of complicating pleuritis from the early stage to the fibrino-fibrous stage, the appearance rate of 3H-thymidine labeled and activated mesothelial cells in the DNA synthetic phase changed quite differently in both effusions. The differences were thought to be due to neoplastic invasion itself or inflammatory reactions to the neoplastic growth.

Adult↗

Cytokinetic variations during ageing and regenerative growth in the jb-1 ascites tumour studied by impulse cytophotometry.

The variation in the DNA distributions in the murine JB-1 ascites tumour was investigated by means of impulse cytophotometry (ICP) on days 2, 4, 7, 10 and 14 after i.p. inoculation of 2.5 times 10-6 tumour cells and on days 1, 2, 3 and 4 after aspiration of the main part of 10-day plateau tumours. The study showed that a considerable fraction of cells with a DNA content of about 2 and 4 n (p smaller than 0.001) accumulated from day 2 to 10. During the same period the fraction of cells in S decreased significantly (p smaller than 0.001). One and two days after aspiration, a rapid rearrangement in the distribution pattern occurred, leading to a predominance of cells with S DNA content, while the fraction of cells around 2 n and 4 n decreased. Three and four days after aspiration the cells again accumulated around the 2 and 4 n values while the fraction of cells with S DNA content decreased. The ICP results were in agreement with our data obtained by autoradiography and Feulgen microspectrophotometry, and ICP is recommended as a rapid and statistically precise method in cytokinetic analysis.

Animals↗

Escherichia coli growth studied by dual-parameter flow cytophotometry.

The growth of Escherichia coli cells has been analyzed for the first time by dual-parameter flow cytophotometry, in which the deoxyribonucleic acid and protein contents of single bacteria have been measured simultaneously with an accuracy of a few percent and at a rate of 3,000 cells/s.

Bacterial Proteins↗

Applications of microcomputer technology to cytophotometry.

Lack of appropriate software support for microprocessor program development has previously limited the applications of such technology in the field of microspectrophotometry. This paper describes our use of a Vickers M86 integrating microdensitometer coupled through a custom-designed interface circuit to a Processor Technology SOL III microcomputer. W e have developed a series of interactive, user-oriented programs for DNA-Feulgen cytophotometry with this instrument to allow automatic storage of data in files on floppy disks and instant retrieval of sets of measurements for statistical processing. The same data files can also be used to generate graphic displays in the form of bar histograms or plots of linear regressions on a video monitor and to produce hardcopy output of data files and graphic displays through the use of a high speed DIABLO printer.

Animals↗