D. Haag

The Proliferative Index (PI) of Human Breast Cancer as Obtained by Flow Cytometry

As known from previous reports, the DNA synthesis fraction of mammary carcinoma cells is correlated with the course of the disease and response to adjuvant therapy. Quantitative parameters of the proliferative activity can be determined by the classic 3HTdR-labelling technique as well as by the more rapid flow cytometric (FCM) DNA analysis. The values of DNA-cytometric S-phase fractions in breast cancers reported up to now were consistently higher than those obtained by the 3HTdR labelling index (LI). This discrepancy was surmounted in the present study by corrections for systemic errors of the method. The fractions of cells in stages of the cell cycle as well as the DNA indices (DI) in 155 cases of primary resectable breast cancers were analyzed by DNA flow cytometry. The patients were aged between 24 and 88 years. As indicated by a bimodal age distribution with a decrease at age 60, the patient population was a representative of the generally known breast cancer incidence. 54% of the patients had aneuploid tumor cells with preferences of DNA indices 1.6 and 2.0. In the remaining 46%, no cell populations with deviating DNA content could be detected, in part possibly due to very small differences beyond the limits of detection. No correlations were found between age, menopausal status, histologic type of tumors, tumor size, fractions in stages of the cell cycle and proliferative index (PI = S + G2 + M in %), except a significant correlation between the S-phase fractions and the (G2 + M)-phase fractions in a ratio of approximately 1.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA-flow-cytometric measurements on the normal, atrophic, hyperplastic and neoplastic human endometrium

DNA distribution patterns and the fractions of the cell cycle phases were determined by means of flow-through cytometry in 87 samples of normal, atrophic, hyperplastic and carcinomatous human endometrium. The S-phase fractions vary during the normal menstrual cycle between 1 and 3% and reach a periovulatory maximum between 4.4 and 4.7%. Atrophic endometrium and regressive-glandular cystic hyperplasia have little DNA synthesis (1.01% and 1.68% S-phase fractions respectively). Proliferating glandular cystic hyperplasia reveals 3.38% S-phase fraction, whereas adenomatous hyperplasia has an increased number of DNA-synthesizing cells (4.81%). The well-differentiated endometrial carcinoma shows no cytophotometrically detectable differences in comparison to adenomatous hyperplasia. All endometrial samples except for poorly differentiated endometrial carcinoma showed a diploid to tetraploid DNA distribution pattern. The poorly differentiated endometrial carcinoma displays two different types: one rapidly growing diploid-tetraploid tumor with 8.0 to 9.6% S-phase fractions, and another type with stemline deviations, polyploid nuclei and less pronounced synthetic activity.

Maligner Granularzelltumor des Großhirnmarkes

SummaryThis is a report of the second known case of a primary malignant granular-cell tumor of the cerebral hemispherical white matter. Two cell types may be distinguished apparently representing different developmental stages of otherwise identical tumor cells. Quantitative histochemical and biochemical studies have shown that tumor cells were containing markedly elevated levels of DNA and RNA. Only few ribosomes and polysomes could be detected, however, by electron microscopy. While cytophotometry disclosed only slightly elevated cytoplasmic proteins, there was a distinct increase in total protein concentration of tumor tissue per unit weight, as measured by conventional techniques. It is suggested that this increase is mainly attributable to structural proteins of cellular and subcellular elements of multiplicating malignant cells. A ratio of RNA to DNA in excess of 1 was found for white matter derived from the central tumor, while the ratios of control tissue were lowered to values far below one due to postmortem changes greatly reducing tissue concentrations of RNA. No detailed characterization of RNA was attempted. The nuclear DNA content of tumor cells reached values equivalent to chromosomal hexadekaploidy. This was in sharp contrast to control data from an Abrikossoff tumor of the oral cavity and from a neurohypophyseal tumorette (“choristoma”), respectively, displaying diploidy only associated with a much lesser increase of cytoplasmic RNA and proteins. Qualitative lipid studies were consistent with a marked active demyelination of the tumor centre as indicated by a severe reduction of cerebrosides and sulfatides and the presence of cholesterol esters. In addition there was a striking loss of phosphatidylethanolamine and a lesser one of sphingomyelin of white matter of both the tumor-stricken and the contralateral unaffected hemispherical regions, possibly suggesting a generally disturbed metabolism of myelin. It is not clear whether these general changes were resulting from the presence of the unilateral tumor or from precocious cerebral involution.

Isolation of single cell nuclei from human epidermis for cytophotometric DNA--measurements.

SummaryDiluted acetic acid was applied to dissociate human epidermis into its constituents. A simple procedure to gain isolated single cells for cytophometric determination of their nuclear DNA content is described. The method is suited for single cell cytophotometric measurements and for cytofluorimetric flow systems likewise. The losses of Feulgen—stainable nuclear DNA during preparation were studied in dependence on the incubation time of the cells in acetic acid. The kinetics of extraction of nuclear DNA by diluted acetic acid follow a reaction of the first order. It was possible therefore to formulate a simple mathematical correction for the losses during the isolation of epidermal cells. Avoiding the disadvantage of cut nuclei in histological sections the method is used to study the DNA histograms of resting, regenerating and pathologically altered human epidermis.ZusammenfassungZur Auflösung menschlicher Epidermis in ihre zellulären Bestandteile erwies sich verdünnte Essigsäure als geeignet. Es wird eine einfache Prozedur zur Gewinnung von Einzelzellen für cytophotometrische Messungen des DNS-Gehaltes in den Zellkernen beschrieben. Diese Methode eignet sich gleichermaßen zur Herstellung von Zellsuspensionen für die Durchflußcytophotometrie wie auch von Ausstrichen auf Objektträger für die Einzelzell-Cytophotometrie. Die Verluste Feulgen-färbbarer Substanz während der Präparation wurden quantitativ erfaßt. Die Extraktion von DNS aus den Zellkernen durch verdünnte Essigsäure folgt näherungsweise einer Kinetik erster Ordnung. Demzufolge kann eine einfache Korrekturformel für die DNS-Verluste während der Präparation aufgestellt werden. Die Methode dient zur Untersuchung der DNS-Histogramme in der normal regenerierenden sowie der pathologisch veränderten menschlichen Epidermis, wobei die Nachteile angeschnittener Zellkerne in histologischen Schnitten weitgehend umgangen werden.

Simultaneous differential staining of nucleic acids and proteins in histological tissues by means of J-Band effect

SummaryA cationic carbocyanine dye, originally used in gel-electrophoresis, stains simultaneously DNA, RNA and proteins also in histological slides or smears. The resulting different colors are demonstrated by various spectral absorbance curves in cell nuclei and cytoplasm. The simple technique is suited also for quantitative cytophotometric determinations.

Ein bisher kaum bekanntes gliogenes Melanin des Gehirns („Cerebellares Gliamelanin“)

Melanosis of the dentate nucleus is an extremely rare condition in which pigment is formed within the cytoplasm and cytoplasmic processes of astrocytes in the dentate nucleus and the cerebellar granular cell layer. The pigment occurs as irregular polygonal structures of rather homogeneous appearance unlike granular neuromelanin located within neurons of the substantia nigra. The diameter of these pigment masses may reach 30 Μm or more. Their absorption of visible light increases steadily towards the shorter wavelengths, and the material is virtually opaque in the violet and ultraviolet region, resembling melanin in this respect. Since the absorption spectrum described by Rabl exhibited a small peak at 525 nm, he regarded the pigment as the hemoglobin derivative pentdyopent. We were unable, however, to confirm this observation. On X-ray diffraction the pigment produced a sharp reflex at 4.93 å as did synthetic melanin prepared from dopamine (but not melanin prepared from serotonin). These observations supported the assumption made by others based on histochemical evidence, that the pigment in the dentate nucleus may belong to the group of melanins. Presumably the glial melanin somehow arises from the metabolic pathway leading to DOPA. An astrocytic site of melanin formation other than in the cerebellum has not been previously described, and this melanin differs in appearance from the granular neuromelanin found in neurons in the substantia nigra. Thus “cerebellar glial melanin” contrasts with neuromelanin of the substantia nigra both in its site of formation and in its morphology. The underlying cause of the glial melanin formation in the cerebellum is not known. There are six cases in the world literature, 5 females and 1 male, ranging in age from 58 to 103 years. Involutional alterations in metabolism related to endocrine factors may possibly play an important part. To the best of our knowledge cerebellar melanosis is of no clinical relevance.

The influence of non-cytotoxic concentrations of the herbicide 2,4-dichlorophenoxyacetic acid on the DNA synthesis in cultured vertebrate cells

The influence of the herbicide 2,4-Dichlorophenoxyacetic acid on the growth of cultured chicken muscle cells was studied by observing morphological changes and by cytophotometric measurement of the DNA content of individual cells. DNA-synthesis curves were derived from the distribution of DNA values. No marked effects were observed at a concentration of 60 μg per liter. In contrast, doses of 600 μg per liter caused a loss in cell differentiation consisting of a partial lack of polar orientation, diminished formation of fibers, and an increase of the N/C ratio. The nuclear matrix of desoxyribonucleoprotein (DNP) changed to a more heteropycnotic structure. Isolated cell necroses were found after 44 hrs of exposure. When compared to the controls, the relative DNA synthesis time was found to be prolonged 1.5-fold. The number of cells in the G2 phase decreased during 44 hrs of exposure. It is concluded from this results that the dosedependent growth inhibition of 2,4-D on vertebrate cells in culture takes effect during DNA synthesis. The morphological changes of the nuclear structure suggest that 2,4-D directly affects the template activity of the DNP matrix.ZusammenfassungDer Einfluß des Herbicides 2,4-Dichlorphenoxyessigsäure auf das Wachstum von Skelettmuskelzellen des Huhns in Zellkulturen wurde anhand morphologischer Veränderungen und durch cytophotometrische Bestimmung des DNS-Gehaltes von Zellkernen untersucht. Aus der Verteilung der DNS-Meßwerte wurden Synthesekurven abgeleitet. Bis zu Konzentrationen von 60 μg/ml waren keine deutlichen Effekte erkennbar. Dagegen verursachten Konzentrationen von 600 μg/ml einen Verlust der Zelldifferenzierung, bestehend in einem Fehlen der polaren Wachstumsrichtung der Zellen, verminderter Faserbildung und einem Ansteigen der Kern-Plasma-Relation. Damit war eine Heterochromatisierung des Desoxyribonucleoproteins in den Zellkernen verbunden. Nach 44stündiger Einwirkung traten vereinzelte Zellnekrosen auf. Die relative DNS-Synthesedauer war gegenüber den Kontrollen auf das 1,5fache verlängert. Die Anzahl der Zellen in der G2-Phase war nach 44 Std Einwirkungsdauer vermindert. Aus den Befunden wird auf eine dosisabhängige Wachstumshemmung von Vertebraten-Zellen durch 2,4-D geschlossen, die während der DNS-Synthese wirksam ist. Die morphologischen Veränderungen der Kernmatrix legen die Vermutung nahe, daß durch 2,4-D die Aktivität des Chromatins direkt beeinflußt wird.

Cytophotometrische Untersuchungen an Zellkernen von experimentell erzeugten Neoplasmen@@@Cytophotometric investigations of cell nuclei from experimentally induced neoplasms

8000 cell nuclei from normal, initiated, promoved and also neoplastically transformed hypo- and hyperchromic nuclei were measured cytophotometrically. Under experimental carcinogenesis there is not any measurable change in the DNA content of the nuclei during the initiation phase. Carcinogenesis of the epidermis induced by 3.4 benzopyrene caused only in the advanced stages of malignancy an increased aneuploidy. In contrast to the above, the diethylnitrosaMine-induced hepatomas showed no changes in respect to the normal values at all. But the correlation between DNA content and nuclear volume is descreased. This is defined by the formula DNA content = average DNA concentration × nuclear volume + const. This was observed in all the investigated cases of neoplastic growth, in contrast to the normal values. A decrease was also observed in carcinogen-initiated tissues which were not visibly altered. The promoting cocarcinogenic phorbol ester A-1 did not change the correlation in contrast to the carcinogens. The decrease in the correlation between nuclear volume and DNA is the first measurable indicator of the altered cell function. This means that there is irreversible damage of the nuclear metabolism caused by changes of the nuclear membrane by carcinogens. Durch Cytophotometrische Messungen an 8000 Zellkernen von normalen, initiierten bzw. promovierten und auch tumorös umgewandelten, hypo- und hyperchromen Zellkernen wird gezeigt, daß während der experimentellen Carcinogenese zunächst keine meßbaren Änderungen des DNS-Gehaltes auftreten. Während bei der Carcinogenese der Epidermis durch 3,4 Benzpyren erst in den ausgeprägteren Erscheinungsformen mit zunehmender Malignitätsstufe auch eine zunehmende Aneuploidie der Zellkerne beobachtet wird, bleibt der durchschnittliche DNS-Gehalt in Zellkernen von experimentell durch Diäthylnitrosamin erzeugten Hepatomen gegenüber Normalwerten unverändert. Dagegen ist die Korrelation zwischen DNS-Gehalt und Zellkernvolumen vermindert, die sich durch stochastische Funktionen der Form DNS-Gehalt = mittlere DNS-Dichte mal Kernvolumen + konst. beschreiben läßt. Dies wurde bei allen bisher untersuchten Fällen von neoplastischem Wachstum im Vergleich zu Normalwerten beobachtet. Die Verminderung ist bereits an carcinogenbeeinflußten (initiierten) jedoch histologisch noch unauffälligen Gewebspartien zu beobachten. Durch den cocarcinogenen (promovierenden) hyperplasiogenen Phorbolester A-1 wird die Korrelation im Gegensatz zu Carcinogenen nicht beeinflußt. Die Verminderung der Kernvolumen-DNS-Korrelation kann somit als erster meßbarer Indicator einer geänderten Zellfunktion gelten und wird als irreversible Schädigung des Zellkernstoffwechsels infolge Veränderungen der Kernmembranen durch Carcinogene gedeutet.

Über die Eignung von Ethidiumbromid als Fluorochrom zur quantitativen Darstellung von Nukleinsäuren in histologischen Präparaten

SummaryIn order to test Ethidiumbromide1 as a marker for DNA in cells, slides with sections of human epidermis and smears of liver cells were stained in a buffered solution of this substance. The staining results demonstrated by photographs indicate, that Ethidiumbromide does not bind selectively to nucleic acids. As a consequence of the spectral properties of the stained tissue elements this fluorochrome does not seem to be suited for quantitative determinations of nucleic acids by measurements of the fluorescence intensity.ZusammenfassungMit dem in der Impulscytophotometrie zu DNS-Messungen benutzten Fluorochrom Ethidiumbromid1 wurden Schnittpräparate menschlicher Epidermis und Tupfpräparate menschlicher Leberzellen behandelt. Die an Fluoreszenz- und Durchlichtphotographien demonstrierten Färberesultate zeigen, daß keine nukleinsäurespezifische Bindung des Farbstoffes erfolgt. Aufgrund der spektralen Eigenschaften der mit dem Fluorochrom tingierten Zellelemente erscheint eine quantitative mikrofluorometrische Substanzmengen-bestimmung nur bedingt möglich.

Cytophotometrische Befunde in der Trachealschleimhaut und in DÄNA-behandelten Trachealpapillomen beim syrischen Goldhamster

Three thousand seven hundred normal appearing nuclei of ten tracheal papillomata, which had been induced in Syrian golden hamsters by DENA, were studied cytophotometrically. Although the nuclei of the papillomata had almost the same content of DNA as did normal cells, they were about twice as large. They were slightly more common in the hyperdiploid range. The mean concentration of the Feulgen-stained DNA per nucleus in the papillomata was pronouncedly decreased. Similar relationships were obtained for the total content of nucleic acids and histones. In addition, the linear regression analysis disclosed for the nuclei of the papillomata a diminished correlation between nuclear volume and content of nucleic acids or histones. The importance of the results are discussed in regards to quantitative, cytophotometric criteria of neoplasia. The changes in the nucelar volume, the widening in the distribution curve of the histograms, and the decrease in the correlation of cell parameters are regarded as cytophotometric equivalents of “stages of malignancy” in the sense applied by Robert Rössle. Mittels cytophotometrischer Methoden wurden insgesamt 3700 morphologisch unauffällige und Zellkerne aus 10 Papillomen der Trachealschleimhaut von DÄNA-behandelten syrischen Goldhamstern untersucht. Die Zellkerne aus den Papillomen besaßen bei annähernd gleichem DNS-Gehalt wie die Normalzellen fast doppelt so große Zellkernvolumina; ihr Häufigkeitsmaximum war gerinfügig in den hyperdiploiden Bereich verschoben. Die durchschnittliche Konzentration von Feulgen-DNS pro Zellkern war bei den Papillomen erheblich erniedrigt. Ähnliche Verhältnisse wurden auch für die Gesamtnucleinsäuren und Histone ermittelt. Die lineare Regressions-analyse deckte für die Papillomzellkerne eine verminderte Korrelation zwischen Kernvolumen und Nucleinsäure bzw. Histongehalt auf. Die Bedeutung der erhobenen Befunde wird im Hinblick auf cytophotometrisch objektivierbare Kriterien des neoplastischen Wachstums diskutiert. Änderungen der Zellkernvolumina, Verbreiterung der Verteilungskurve und Verminderung der Korrelation von Zellparametern werden als cytophotometrisch erfaßbare Äquivalente für “Stufen der Malignität” im Sinne von Robert Rössle gedeutet.