Individual Cell Nuclei Research Articles

Microdosimetric concepts in radioimmunotherapy.

In microdosimetry particular emphasis is placed on the stochastic fluctuation of dose in small target volumes such as individual cell nuclei or chromatin fiber, and their relevance to radiobiologic toxicity. Thus microdosimetry is intimately associated with models of radiation action. There are three principal areas where microdosimetry has been applied: (1) radiation protection, (2) high LET radiotherapy, e.g., neutron therapy, and (3) incorporated radionuclides, and in this latter category the importance of microdosimetry to the radiobiology of radiolabeled antibodies is becoming increasingly recognized. The objective of microdosimetry is the complete characterization of energy deposition within all target volumes throughout the tissue of interest. The importance and relevance of this pursuit will depend upon the properties of the radionuclide emissions and the spatial distribution of the radionuclide relative to the target volumes. If the distribution of internal emitters within both malignant and normal tissue is uniform, the application of microdosimetry to radioimmunotherapy (RIT) is limited to alpha-emitters and Auger emitters. Under such circumstances the traditional MIRD formalism for the evaluation of tumor and tissue doses from the commonly used beta-emitters is entirely adequate. This, however, is rarely the case. When the distribution of radiolabeled antibody is nonuniform, techniques of dose averaging over volumes greater in size than the individual target volumes can become inadequate predictors of the biological effect. The concepts, methods, and realm of applicability of microdosimetry within the field of radioimmunotherapy are emphasized in this paper.

Influence of Microdosimetric Quantities on Observed Dose-Response Relationships in Radiation Therapy

The steepness of dose-response curves in radiation therapy depends to a large extent on the statistics of cell killing. This is so if the last few clonogenic tumor cells have to be hit or eradicated by other means to cure the patient. The steepness is dependent on the number of clonogenic cells in the tumor and the possible variation in their sensitivity. However, the uniformity of the dose distribution is also important and a decreased slope may result when the delivery of the dose is nonuniform or statistically uncertain. The variance in the energy imparted at the microdosimetric level to individual cell nuclei constitutes the ultimate limit of the variance in delivered dose at a given mean tumor dose. Considering all dosimetric variances it is shown that for low-LET beams the conventional microdosimetric variance will dominate, while in neutron and high-LET beams in general the microdosimetric variance may contribute significantly to the observed dose-response relationship. As a result the normalized slope of the dose-response curve for tumor control and normal tissue complications with neutrons and other high-LET beams will be reduced compared to that with photons. This conclusion is found to be in quantitative agreement with available data from clinical trials with neutron therapy. Finally, it is pointed out that for beams with a very high RBE and LET it may be favorable to deliver a fraction of the total dose in the form of conventional low-LET radiation. This addition of low-LET radiation may be desirable to ensure a dose to all clonogenic tumor cell nuclei that is sufficiently high and uniform to achieve a high probability of tumor control.

Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization.

Detection and subcellular localization of human immunodeficiency virus (HIV) were investigated using sensitive high-resolution in situ hybridization methodology. Lymphocytes infected with HIV in vitro or in vivo were detected by fluorescence after hybridization with either biotin or digoxigenin-labeled probes. At 12 hr after infection in vitro, a single intense signal appeared in the nuclei of individual cells. Later in infection, when cytoplasmic fluorescence became intense, multiple nuclear foci frequently appeared. The nuclear focus consisted of newly synthesized HIV RNA as shown by hybridization in the absence of denaturation and by susceptibility to RNase and actinomycin D. Virus was detected in patient lymphocytes and it was shown that a singular nuclear focus also characterizes cells infected in vivo. The cell line 8E5/LAV containing one defective integrated provirus revealed a similar focus of nuclear RNA, and the single integrated HIV genome was unequivocally visualized on a D-group chromosome. This demonstrates an extremely sensitive single-cell assay for the presence of a single site of HIV transcription in vitro and in vivo and suggests that it derives from one (or very few) viral genomes per cell. In contrast, productive Epstein-Barr virus infection exhibited many foci of nuclear RNA per cell.

Spatial Distribution Patterns of Energy Deposition and Cellular Radiation Effects in Lung Tissue Following Simulated Exposure to Alpha Particles

Abstract Randomly oriented sections of rat tissue have been digitised to provide the contours of tissue-air interfaces and the locations of individual cell nuclei in the alveolated region of the lung. Sources of alpha particles with varying irradiation geometrics and densities are simulated to compute the resulting random pattern of cellular irradiation, i.e. spatial coordinates, frequency, track length, and energy of traversals by the emitted alpha particles. Probabilities per unit track length, derived from experimental data on in vitro cellular inactivation and transformation, are then applied to the results of the alpha exposure simulations to yield an estimate of the number of both dead and viable transformed cells and their spatial distributions. If lung cancer risk is linearly related to the number of transformed cells, the carcinogenic risk for hot particles is always smaller than that for a uniform nuclide distribution of the same activity.

Evidence of an acquired increase in mitogenesis in streptozotocin-diabetic rats, apparently relating to some tissue factor

We have previously reported that rats which have been suffering from streptozotocin-diabetes for 4 weeks show a supranormal mast cell mediated mitogenesis in mesenteric windows and in the skin; this late emerging, augmented mitogenic responsiveness appears, to be unaffected by insulin per se. To test whether this increased proliferogenic response is effected by some acquired quality within the tissue rather than a systemic factor in the blood, we studied mast cell mediated mitogenesis in organ-cultured intact mesenteric windows from rats with diabetes of 4 weeks' duration, using a biochemically-defined serum-free growth medium. Mast cells were activated by Compound 48/80 and their secretion was quantified biochemically in terms of histamine release. The mast cell-dependent mitogenic reaction in the predominant, morphologically discrete fibroblasts and mesothelial cells was quantified photometrically using Feulgen-absorption analysis of individual cell nuclei, and by determination of the mitotic index. Both types of target cell responded to a significantly greater degree mitogenically in diabetic compared with control tissue. This finding suggests that a considerable part of the increased mitogenic responsiveness previously observed in diabetic animals in vivo is causally related to some tissue-bound, i.e., cellular and/or extracellular factor(s) acquired during the course of the disease.

In vivo cell kinetic effects of cis-platinum on human ovarian cancer xenografts measured by dual parameter flow cytometry.

Dual parameter flow cytometry, measuring DNA and nuclear protein of individual cell nuclei simultaneously, makes it possible to follow cell kinetic perturbations in six distinct compartments of the cell cycle following chemotherapy in vivo. Human ovarian cancer xenografts in nude mice from a primary and recurrent tumor of the same patient were studied. The response to intraperitoneal application of cis-platinum was assessed by tumor volume measurements, changes in labeling indices by autoradiography, and dual parameter flow cytometry. Sequential tissue samples were taken from each tumor using fine needle aspirations as a microbiopsy method. Pretherapy samples were compared to multiple specimens collected up to 18 days after therapy. Morphologic changes of each specimen were also assessed. Cis-platinum affects malignant cells in the G1B, S, G2A, and G2B compartments with various intensities and different time frames, depending on the drug sensitivity of each individual tumor.

Accurate determination of DNA content in single cell nuclei stained with Hoechst 33258 fluorochrome at high salt concentration

In an attempt to achieve accurate quantification of DNA levels in cell nuclei, we studied the influence of salt concentration on the fluorescence of cell nuclei complexed with Hoechst-33258 (Hoe) fluorochrome. The fluorescence of cell nuclei was compared with that of extracted DNA as well as that of nucleosome core. Conformational changes in these complexes were examined by measuring both fluorescence anisotropy and fluorescence lifetime in the nanosecond region. The results showed that the fluorescence of DNA-Hoe was quenched by the nucleosomal structure, there being an associated increase in anisotropy and a decrease in the fluorescence lifetime; however, the fluorescence was restored to the original level by the addition of a high concentration of NaCl, CsCl, or LiCl. The reduction in fluorescence may have been due to loss of fluorescence energy caused by collision of the fluorophore with histones in the nucleosome. The addition of 1 M NaCl to the medium used for staining with Hoe greatly stabilized the fluorescence of DNA in cell nuclei. The DNA content of individual cell nuclei was determined by comparing the fluorescence of these nuclei with that of a standard DNA solution. For lymphocytes and liver ploidy cells, reasonably accurate values were obtained by applying the present method.

Regulation of pro-opiomelanocortin gene transcription in individual cell nuclei.

A nonrepetitive complementary RNA probe specific for an intervening sequence of the rat pro-opiomelanocortin (POMC) gene primary transcript was used to analyze the hormonal regulation of POMC gene transcription in individual cell nuclei in the rat pituitary by in situ hybridization. This probe recognized only full-length POMC heterogeneous nuclear RNA, as verified by Northern blots of pituitary RNA. When pituitary sections were hybridized with this 3H-labeled POMC intron A probe, silver grains were predominantly localized over the nuclei of cells that expressed POMC in the anterior and intermediate lobes. Adrenalectomy increased both the average grain density over corticotroph nuclei and the number of cells in the anterior pituitary with significant numbers of silver grains over their nucleus. Dexamethasone administration to intact or adrenalectomized rats results in the rapid (within 30 minutes) disappearance of silver grains over the nuclei of corticotrophs in the anterior lobe, suggesting that POMC gene transcription had been inhibited. However, adrenalectomy or dexamethasone administration did not alter the silver grain density over nuclei of intermediate lobe melanotrophs. Thus, this in situ hybridization assay utilizing an intervening sequence-specific POMC probe can measure rapid physiological changes in POMC heterogeneous nuclear RNA in individual cell nuclei.

Annual cycle of the Sertoli cell population in adult stallions.

Stereological methods were employed in two experiments with adult stallions: to confirm seasonal variation in number of Sertoli cells and to characterize the annual cycle of the Sertoli cell population. In the first experiment, testes from 28 adult (4-20 years old) horses obtained in the non-breeding season (December-January) were compared to testes from 28 adult horses in the breeding season (June-July). Sertoli cell numbers were calculated from the nuclear volume density, parenchymal volume, and volume of an individual Sertoli cell nucleus determined by reconstruction of serial sections or from average height and width measurements. The number of Sertoli cells per testis was significantly greater in the breeding season. In a second experiment involving 43-48 adult horses in each 3-month period, the Sertoli cell population was higher (P less than 0.05) in May-July than other periods and higher (P less than 0.01) than in November-January. These combined studies confirm seasonal differences in the Sertoli cell numbers per testis and define the annual cycle of the Sertoli cell population in adult stallions.

Cytogenetic evidence of inducible processes linked with metabolism of a xenobiotic chemical in adult and larval Mytilus edulis

Sister chromatid exchange (SCE), a sensitive cytogenetic endpoint for measuring the effects of mutagens on chromosomes, was assayed in vivo in adult and larval mussels to determine whether these different life-history stages possess any capability for converting promutagens to mutagenic species. Cyclophosphamide (CPA), a water-soluble promutagen that is dependent for its mutagenic activity in mammals on transformations accomplished via the cytochrome P-450 pathway, was selected for this investigation. CPA was found to cause increased frequencies of SCE in both adult and larval M. edulis. In addition, the presence of phenobarbital (PB), an inducer of the microsomal detoxication system in mammals, was found to increase the levels of SCE produced by CPA, indicating that the effect of PB may have been to increase the rate at which CPA was metabolized. Operating as it does at the level of the individual cell nucleus, SCE is thus shown to have potential as an extremely sensitive indicator of stimulation of the microsomal detoxication system in the common mussel. The apparent inducibility of this activity may have important consequences for these organisms when they are exposed to the complex mixtures of xenobiotic chemicals found in the environment.

DNA distribution, cytosol estrogen receptors and axillary nodes as prognostic predictors in breast carcinoma.

One hundred and fifty patients with breast carcinoma were examined to compare axillary node status, estrogen receptor level and cellular DNA content as prognostic indicators. Seventy-four per cent of the patients were postmenopausal and forty per cent had axillary node metastases. Estrogen receptor was measured by isoelectric focusing in polyacrylamide gel. DNA was measured in individual cell nuclei by means of Feulgen-acriflavine-sulphate stained imprints. Fifty-two per cent of the tumors had diploid and/or tetraploid DNA pattern, and the rest aneuploid pattern. Axillary node metastases, aneuploid DNA pattern and low level of estrogen receptor were related to recurrence. When introduced into Cox's proportional hazards procedure, axillary nodes and estrogen receptor level but not DNA pattern remained as significant predictors of recurrence.

Polyploid nuclei in human artery wall smooth muscle cells.

Although polyploid nuclei have long been known to be present in many adult human tissues, the ploidy of smooth muscle cells in human artery wall has never been determined. We measured DNA content in individual smooth muscle cell nuclei of artery wall specimens by two means: Feulgen microdensitometry and flow microfluorimetry. A significant percentage of nuclei were polyploid; most of these were tetraploid, although higher levels were also found. The frequency of polyploidy varied with age from less than 1% at birth to a mean of 7% in adult aortic, carotid, and iliac vessels. Atherosclerotic plaques had a lower tetraploid content than the underlying media, whereas normal intima was similar to the corresponding media. The increase in frequency of hyperploid smooth muscle cell nuclei correlates with the normal growth, development, and aging of human artery wall. We suggest that the regular existence of a subset of polyploid smooth muscle cells may indicate an important functional role for this phenotype.

DNS-Gehalt and Zellzahl in Herz and Leber von Kindern: Vergleichende biochemische, cytophotometrische and histologische Untersuchungen

Summary In order to elucidate whether and when and age-dependant polyploidization process takes place in the course of normal growth of the organ, analogous to that in the myocardium, the DNA content was studied in 26 livers and in 24 hearts. Human material between the ages of the 28th week of gestation and the 17th year of life were employed. The organs were weighed and prepared within 60 hours after death. DNA content was determined in the hearts and livers using the diphenylamine reaction according to Dische and Burton. Feulgen cytophotometric measurements obtained the DNA content of individual cell nuclei and the various ploidy levels of the tissue. Using the quantitative relationship of connective tissue to parenchymal tissue nuclei obtained histologically and the corresponding organ weight, the absolute cell number in the organ was computed. These studies led to the following results: 1. The biochemically determined DNA concentration in the liver is more than 2.0 mg/g tissue and decreases to approximately 1.0 mg/g at birth, after which it remains at this level as a constant until the 17th year of life. 2. The total amount of DNA in the liver increases from 50 mg at the end of the fetal period to over 1500 mg at the age of 17 years. 3. Over 90% of the parenchymal cells in the liver are diploid until the 8th year of life, after which more than 10% tetraploid cells appear, whose proportion increases to at most 15% up to the 15th year of life. Higher ploidy level cells cannot be found in morphologically unremarkable organs. 4. The total cell count in the liver increases from 20 × 10 9 cells at birth to approximately 70 × 10 9 cells at 6 years old; at age 15 years 180 × 10 9 cells are present in the liver. Mesenchymal and parenchymal cells increase in parallel during this time. The number of parenchymal cells amounts to 10 × 10 9 at birth and 110 × 10 9 at the age of 15 years. Although functional loading appears to be involved as a stimulus for polyploidization in the heart, many observations point to the idea that the ploidy level in the liver is hormone-dependent. This idea is supported by our observations of a case of adrenal cortical atrophy with ploidy level in the hepatocytes which was elevated above normal.

Correlative Electron Microscopic and Cytofluorometric Study of Chromatin Conformation in Human Leukemia Cells

In order to establish a sensitive probe to analyze and interpret the changes in chromatin conformation expressed in individual leukemic cell nuclei we have correlated data from a concomitant ultrastructural and cytofluorometric study on high blast count leukemic bone marrows utilizing the nuclear probe and metachromatic fluorochrome, acridine orange. At the electron microscopic level, the incorporation of this dye into “active euchro- matin” of morphologically identifiable leukemic blasts is visualized as Frenster’s1 electron dense reaction product (Fig 1). Cytofluorometrically, in the absence of RNA, acridine orange differentially stains double stranded DNA green (F530) and single stranded DNA red (F>600). Darzynkiewicz2 has demonstrated that the sensitivity of DNA in situ to denaturation is higher in quiescent cells than in actively cycling ones.

Estimation of haploid cell content of parthenogenetic turkey embryos: A cytofluorometric study

AbstractA quantitative cytofluorometric study was undertaken to test the proposition that parthenogenetic development in turkeys begins in a haploid cell and proceeds toward the development of a completely diploid embryo. Embryo cell suspensions were obtained from eggs laid by virgin Beltsville Small White (BSW) turkey hens, and the cells from individual embryos were fluorescently stained with acriflavine, which is specific for DNA. The fluorescent emissions from individual cell nuclei were analyzed on a modified Bio/Physics Cytofluorograf, Model 6300A, and DNA distributions were obtained by analyzing nearly all of the cells in embryos ranging in age from five to nine days. These distributions were compared by computer analysis to the DNA distribution of a normally produced embryo. The DNA distributions revealed that many embryos contained large proportions of haploid cells and other abnormal cells. This study of haploid cells lends further support to the proposition that parthenogenesis in turkeys originates in a haploid egg cell, and diploidization observed in embryos occurs after meiosis. It was also shown that proportions of haploid and other non‐diploid cells declined with increasing embryo age.

Nuclear DNA content of the normal and hypertrophied myocardium in the pediatric age group

The DNA content of individual muscle cell nuclei in normal and hypertrophied children's hearts was studied by microspectrophotometric measurements of Feulgen-stained sections. Normal newborn myocardium was found to have a diploid DNA distribution. In four patients (ranging in age from 2 years, 10 months to 12 years) with cardiac hypertrophy, a polyploid distribution of DNA values was found. This finding is consistent with the findings in hypertrophied adult hearts. Polyploidization in both adults and children may occur in response to the demands of higher cellular activity in the heart. The mechanism by which increased functional requirements induce polyploidy in the human myocardium is unknown.

Increase in viral antigen in individual polyoma-virus-infected mouse kidney cells as studied by quantiative immunofluorescence.

AbstractPolyoma‐virus infection of mouse kidney cells in vitro was studied on the single cell level by quantitative cytochemical techniques. Cells were fixed in acetone at different times after infection and reacted with fluorescein‐conjugated mouse anti‐polyoma‐virus immune serum. Visual determinations were made as to whether the cells were fluorescent (“positive”) or not (“negative”). Visually the fluorescence was located in the nuclei in the “positive” cells. The fluorescence intensities of individual cell nuclei were then measured in a microspectrofluorimeter. Nuclei with considerable fluorescence intensity were noted to be visually “positive”. However, nuclei of cells which were usually judged to be “doubtful” could also be shown to have measurably greater fluorescence intensity than control non‐infected cells. Increase in fluorescence values were noted 6‐11 h after infection whereas hemagglutination tests did not become positive until 10‐20 h later. With increasing time post infection, the fraction of fluorescence positive cells increased both as judged visually and as determined by the microfluorimeter. The variation in fluorescence intensities among different nuclei also increased and, 48‐50 h after infection, the ratio between the highest and the lowest fluorescence values of positive cells was about 10:1. Higher fluorescence intensities were found in cytoplasms of cells with “positive” nuclei than in cells with “negative” nuclei. The cytoplasmic and nuclear fluorescence intensities correlated well in cells with “positive nuclei”. The polyoma‐virus infection‐induced stimulation of DNA synthesis was demonstrated by increased incorporation of 3H‐thymidine and by increased amounts of DNA in the cells as measured by Feulgen microspectrophotometry. The amount of DNase‐resistant DNA (i.e. viral DNA) correlated well with the intensity of immunofluorescence measured on the same individual nuclei of infected cells. This was taken as one indication that viral antigens can be measured on the single cell level by quantitative immunofluorimetry.

The hypothalamic-hypophysial neurosecretory system in the iguanid lizard, Dipsosaurus dorsalis: A qualitative and quantitative study

The neurosecretory system of the desert iguanid Dipsosaurus dorsalis consist of paired paraventricular and supraoptic nuclei from which four separate neurosecretory tracts arise and coalesce to form a common tract that runs through the median eminence region, where it comes into intimate contact with capillaries of the portal system, to the pars nervosa. Herring bodies were observed in both the median eminence and in the pars nervosa. In addition paired tracts from the paraventricular nuclei pass directly to the pars nervosa via a dorsal route without entering the median eminence. The paraventricular and supraoptic nuclei are connected at their anterior and posterior margins by bridges of neurosecretory cells. The numbers of neurosecretory cells, the dimensions of the hypothalamic centers and of individual cell nuclei were counted and measured. Round and oval cell nuclei were identified in both supraoptic and paraventricular areas, the former predominating in the paraventricular nuclei and the latter in the supraoptic nuclei.

Cytochemical evidence of nucleoprotein changes in rat adrenal cortex following hypophysectomy or dexamethasone suppression

Changes in the characteristics of nuclear chromatin of rat adrenocortical cells were studied following withdrawal of ACTH stimulation by hypophysectomy or by dexamethasone suppression. The binding of acridine orange to individual cell nuclei was measured by microfluorimetry. Both hypophysectomy and dexamethasone suppression reduced the binding of acridine orange to nuclear chromatin to 40–70% of the controls. Simultaneously, the thermal stability of the deoxyribonucleoprotein complex increased significantly. The chromatin changes observed seem to reflect the situation in vivo, bearing relation to the functional activity of the deoxyribonucleoprotein complex.

Recording melting and renaturation profiles of the DNP complex of individual cell nuclei by a microfluorometric method using acridine orange

Recording melting and renaturation profiles of the DNP complex of individual cell nuclei by a microfluorometric method using acridine orange