Immunocytochemical demonstration and morphometric study by image analysis of estrogen receptors in carcinoma of the breast: study of 30 cases

The method described associates on cytological preparations, immunocytochemical reaction, counter-staining, random and morphometrical study by image analysis. It permits the quantification of estrophilin in breast carcinoma and the estimation of the heterogeneity of the tumour staining.     

Ki-67 labeling in postmitotic cells defines different Ki-67 pathways within the 2c compartment

Simultaneous quantification of DNA and Ki-67 proliferation-associated antigen was performed using fluorescence image cytometry. In the MCF-7 cell line, the Ki-67 antigen content increases during the cell cycle, and its intranuclear distribution pattern varies. Quantitative evolution of Ki-67 content as a function of nuclear area makes it possible to define several pathways followed by cells going through the 2c compartment. 1) In some cells, the amount of Ki-67 antigen remains constant during G1 (Ki-67 stable pathway), and a characteristic speckled pattern can be observed. 2) In the larger fraction of cells analyzed, there is a postmitotic decrease in the Ki-67 (Ki-67 decrease pathway) content. In this pathway, labeling is located in the nucleoplasm in small nuclei, is located in nucleoli in intermediate-sized nuclei, and is absent from larger nuclei (G0). A progressive increase in Ki-67 content (Ki-67 increase pathway) was observed from intermediate-sized nuclei to S phase nuclei. From these results, we hypothesize that the Ki-67 stable pathway is the G1 phase of newly formed cells going directly to S phase in local optimal conditions of growth and that Ki-67 decrease pathway and Ki-67 increase pathway correspond to cells whose progression to S phase is regulated by extracellular factors.     

Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.

This study is intended to be the first step of an in situ exploration of the intranuclear DNA distribution by image cytometry (SAMBA) with several fluorochromes. The nuclear DNA content and the chromatin pattern, revealed by ten fluorochromes (HO, DAPI, MA, CMA3, OM, QM, AO, EB, PI, and 7-AMD), were analyzed on mouse hepatocytes fixed by the Boehm-Sprenger procedure optimal for preserving the chromatin pattern. The question was whether fluorochromes specific to DNA make it possible to accurately quantitate the total nuclear DNA content when the chromatin pattern is preserved. Only HO and MA were found to provide satisfactory quantitation of nuclear DNA content, as assumed by both a small CV and a 4c to 2c ratio equal to 2. PI, EB, 7-AMD, and OM provided higher CV values, although the 4c to 2 c ratio was still equal to 2. QM, AO, CMA3, and DAPI provided non-reproducible and non-stoichiometric nuclear DNA content measurements under the fixation conditions used. The intranuclear and the internuclear SD of the fluorescence intensities describing the fluorescence pattern of the 2c hepatocytes proved to vary according to both the basepair specificity and the binding mode of the fluorochromes. The results reported here argue in favor of an external binding of 7-AMD to DNA and an increased quantum yield of QM when bound to AT-rich DNA. For PI, EB, 7-AMD, and OM, the measured DNA content increased with the fluorescence distribution heterogeneity. This correlation was not observed with other fluorochromes and is suggested to result from decreased fluorochrome accessibility to DNA when the chromatin is condensed. This study demonstrates that under conditions that preserve chromatin organization, only HO for AT-rich DNA and MA for GC-rich DNA can be used, alone or in combination, to measure nuclear DNA content. With other fluorochromes, either the measured DNA content or the chromatin pattern is assessed in suboptimal conditions when fluorescent image cytometry is used.     

Quantitative analysis of cytoskeletal proteins throughout the cell cycle of the MRC-5 fibroblastic cell line

Fluorescent dyes were used to stain actin, vimentin, tubulin and DNA in the same MRC-5 fibroblastic cells. Cytofluorometry and image analysis were then used to quantitatively evaluate the F actin, vimentin and tubulin content throughout the cell cycle. The results showed that different cells can have the same DNA content while their cytoskeletal protein content is variable. The data also showed that cytoskeletal protein content variations exist throughout the cell cycle of the fibroblastic cell line. The F actin content increased during the cell cycle from G1 to G2 phases and decreased in M phase. The amount of tubulin in the G2 was about twice as much as that in the G1 phase, before decreasing in the M phase; there was a threshold of tubulin content for G2 cells entering S phase.     

A quantitative analysis of the human bone marrow granulocytic cell lineage using the SAMBA 200 cell image processor. I. The normal maturation sequence

A quantitative image analysis of the human normal bone marrow granulocytic line was performed using the SAMBA 200 image analyzer. The steps of image acquisition, preprocessing, segmentation and parametrization are described. Forty-one parameters were computed on 941 cell images belonging to the various maturation stages. The automated classification of these cells based upon a stepwise linear discriminant analysis resulted in 77% correctly classified cells; the five most discriminating parameters were the nuclear area, the nuclear convexity degree, the average cytoplasmic hue, the regularity of the nuclear boundary and the average cytoplasmic luminance. The evolution of the parameters correlates well with the cytologic evolution and the biochemical and functional events during the maturation process. It can be inferred from our results that the maturation sequence can be subdivided into two phases according to the evolution of the cell profiles. The first phase, from myeloblast to myelocyte, is discontinuous and appears as the critical point with regard to the expression of genes. The second phase, from myelocyte to polymorphonuclear cell, is a continuous sequence of transformations leading to the functional granulocyte.     

1-Deoxysphinganine initiates adaptive responses to serine and glycine starvation in cancer cells via proteolysis of sphingosine kinase

Cancer cells may depend on exogenous serine, depletion of which results in slower growth and activation of adaptive metabolic changes. We previously demonstrated that serine and glycine (SG) deprivation causes loss of sphingosine kinase 1 (SK1) in cancer cells, thereby increasing the levels of its lipid substrate, sphingosine (Sph), which mediates several adaptive biological responses. However, the signaling molecules regulating SK1 and Sph levels in response to SG deprivation have yet to be defined. Here, we identify 1-deoxysphinganine (dSA), a noncanonical sphingoid base generated in the absence of serine from the alternative condensation of alanine and palmitoyl CoA by serine palmitoyl transferase, as a proximal mediator of SG deprivation in SK1 loss and Sph level elevation upon SG deprivation in cancer cells. SG starvation increased dSA levels in vitro and in vivo and in turn induced SK1 degradation through a serine palmitoyl transferase-dependent mechanism, thereby increasing Sph levels. Addition of exogenous dSA caused a moderate increase in intracellular reactive oxygen species, which in turn decreased pyruvate kinase PKM2 activity while increasing phosphoglycerate dehydrogenase levels, and thereby promoted serine synthesis. We further showed that increased dSA induces the adaptive cellular and metabolic functions in the response of cells to decreased availability of serine likely by increasing Sph levels. Thus, we conclude that dSA functions as an initial sensor of serine loss, SK1 functions as its direct target, and Sph functions as a downstream effector of cellular and metabolic adaptations. These studies define a previously unrecognized “physiological” nontoxic function for dSA.     

Quantitation of AgNORs by flow versus image cytometry.

AgNORs are nucleolar proteins that interact specifically with silver salts. The size of silver precipitates measured by image analysis (ICM) in cycling cells proved to be inversely proportional to the cell cycle time and provided a significant correlation with prognosis for a large spectrum of cancers. Because ICM is time-consuming and poorly reproducible among laboratories using different imaging settings, this article presents a new approach to AgNOR quantitation based on flow cytometry (FCM). We report that silver precipitates caused a great decrease in the forward scattered light and that this effect was correlated with the AgNOR's relative area as measured by ICM. These results were confirmed by measuring cell lines having different cell cycle durations. Moreover, double staining using APase-Fast red fluorescence to reveal the Ki-67/MIB 1 antigen of cycling cells and silver nitrate to stain the AgNORs was successfully analyzed by FCM. The procedure makes it possible, for the first time, to validly and rapidly compare the growth fraction and cycling speed of partially proliferating cell populations, such as tumors.     

Relations entre la prolifération et la différenciation cellulaires dans l'intestin embryonnaire et larvaire dePleurodeles waltlii Michah

Résumé Entre la fin du développement embryonnaire (stade 34) et le début de la vie larvaire (stade 37), l'épithélium intestinal du pleurodèle passe en 4 jours de l'état de cordon endodermique indifférencié à celui d'épithélium fonctionnel constitué de cellules absorbantes à plateau strié et de cellules à mucus. Au cours de cette différenciation, le nombre de cellules augmente de 2,6 fois environ bien que l'activité mitotique diminue régulièrement par abaissement du coefficient de prolifération de 52% à 22%. Les mitoses qui, dans le cordon endodermique au stade 34 sont uniformément distribuées, apparaissent brusquement localisées, au stade 36, dans les nids cellulaires sous-épithéliaux qui représentent dès lors la totalité du compartiment générateur intestinal. Avant même d'être localisé, ce compartiment produit autant de cellules qui, bloquées en phase G₁ du cycle mitotique, cessent de se diviser et se différencient, que de cellules qui parcourent le cycle mitotique et se divisent à nouveau. Un tel processus laisse persister dans l'épithélium intestinal un nombre constant de cellules douées d'activité mitotique et capables d'assurer ainsi le renouvellement de ce tissu.L'évolution de la prolifération et de la différenciation cellulaires dans l'intestin de pleurodèle est très comparable à celle observée chez les Mammifères et confirme, en particulier, l'analogie longtemps contestée entre les cryptes de Lieberkühn de ces derniers et les nids cellulaires sous-épithéliaux des Urodèles.

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Relationships between the cell proliferation and the differentiation in the embryonic and larval intestine ofPleurodeles waltlii michah I. Normal development

Summary Differentiation of the intestinal epithelium ofPleurodeles occurs during the last period of embryogenesis (stage 34) and is completed during the first stages of larval development before the onset of feeding (stage 37). In the course of this 4-day period the intestinal epithelium, which is a closed endodermal cylinder at stage 34, becomes a functional epithelium constituted by columnar absorbing cells and goblet cells. During intestinal differentiation, the cell number rises although the growth fraction decreases from 52% to 22%. At stage 34, mitoses are randomly distributed throughout the endoderm, but at stage 36 they become confined to cell nests which appear beneath the epithelium.The cell nests correspond to the proliferating compartment which produces an equal number of dividing cells and of resting cells: these cells are arrested in the G₁ phase of the generative cycle and differentiate. Such a pattern of proliferation and differentiation maintains a constant number of proliferating stem cells which subserve the renewing function in the intestinal epithelium after the onset of feeding. The relationships between cell proliferation and differentiation in the developing intestinal epithelium ofPleurodeles are closely similar to those observed in Mammals and suggest particularly that the intestinal cell nests of Urodela are analogous to the crypts of Lieberkühn in higher Vertebrates.

Ce travail a bénéficié de l'aide du CNRS (ATP N^o A655 1799) et de l'INSERM (AT N^o 74 142036)     

Relations entre la prolifération et la différenciation cellulaires dans l'intestin embryonnaire et larvaire dePleurodeles waltlii Michah

Résumé Les chalones 1 et 2, extraites de l'intestin de pleurodèle adulte, inhibent en phase G₁ et G₂ respectivement les cycles mitotiques de l'épithélium intestinal embryonnaire. Les effets de ces chalones sur la prolifération et la différenciation cellulaires dans ce tissu ont été étudiés en fonction de la dose injectée, du stade de développement et de la durée du traitement. L'inhibition provoquée par la chalone 2 est proportionnelle à la dose injectée entre deux seuils de concentration. Le quart environ des cellules intestinales en activité mitotique est insensible à la chalone 2 même à la suite d'injections répétées de l'inhibiteur. Seules les cellules intestinales des embryons âgés (stade 34) sont sensibles à cette chalone et répondent par un allongement de la phase G₂ qui, malgré des injections répétées de l'inhibiteur, n'excède pas une vingtaine d'heures. La sensibilité des cellules de l'épithélium intestinal à la chalone 1 se manifeste à la fin du développement (stade 33), comme dans le cas de la chalone 2. A l'égard de la chalone 1, la population cellulaire en activité mitotique dans l'intestin embryonnaire apparaît hétérogène et comprend: 50% de cellules aptes à être inhibées par des doses faibles de chalone 1; 25% de celludes aptes à n'être inhibées que par des doses de chalone 1 cent fois plus élevées et 25% environ de cellules insensibles à cet inhibiteur. Les injections répétées de chalone 1 bloquent définitivement en phase G₁ la moitié environ des cellules en activité mitotique dans l'épithélium intestinal indifférencié au stade 34; en outre, elles accélèrent la consommation du vitellus, favorisent la différenciation des cellules à mucus et diminuent le nombre des cellules constituant les nids sous-épithéliaux qui apparaissent au stade 36 et représentent le compartiment générateur de l'épithélium intestinal. Les réultats obtenus permettent de proposer un modèle de cinétique de la prolifération cellulaire au cours de la différenciation de l'épithélium intestinal du pleurodèle; de plus, ils conduisent à l'hypothèse que le nombre de divisions subies par une cellule embryonnaire et le taux de chalone dans le tissu auquel elle appartient, sont les deux signaux complémentaires qui déclenchent le blocage du cycle mitotique et l'achèvement de la différenciation dans cette cellule.

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Relationships between the cell proliferation and the differentiation in the embryonic and larval intestine ofpleurodeles waltlii michah. II. Effects of intestinal chalones extracted from the intestine of the adult newt

Summary The intestinal chalones 1 and 2, extracted from the intestine of the adult newt, are known to inhibit the G₁ and G₂ phases of the cell cycle in the embryonic intestine. The effects of these intestinal chalones on the proliferation and differentiation of intestinal cells of newt embryos were studied with special attention to the dose-response relationship, the embryonic stage and the duration of treatment. The chalone 2 triggered a linear, dose-dependent inhibition between two concentration thresholds; nevertheless about 25% of the cycling cells were not inhibited either by the highest doses injected or by repeated injections. Sensitivity to chalone 2 appeared in the intestinal epithelium at the end of embryonic development (stage 34) but the cells were not delayed in the G₂ phase for more than about 20 h in spite of repeated injections. It was inferred from the doseresponse curve of the mitotic inhibition by chalone 1, that the intestinal cell population was heterogeneous: about 50% of the cycling cells were inhibited by low concentrations of chalone 1; an additional proportion of about 25% of cycling cells was inhibited by 100 x more concentrated chalone 1 and the remaining 25% was insensitive to the inhibitor. Repeated injections of chalone 1 blocked about 50% of the cycling cells definitively in the G₁ phase, speeded up digestion of yolk platelets, promoted the differentiation of goblet cells and depressed the number of stem cells in the proliferative compartment located beneath the epithelium. A kinetics model of cell proliferation and cell differentiation in the intestinal cell lineages was elaborated and it was suggested that the arrest of mitotic activity and the completion of differentiation in an embryonic cell depends on two incoming signals: one is intracellular and appears when the required number of cell cycles has occured in the cell lineage, leading to a committed stem cell sufficiently differentiated to synthesise chalone and to respond to chalone; the other signal is extracellular and appears when the chalone concentration is high enough: i.e. when the required number of cells is obtained in this tissue.

Ce travail a bénéficié de l'aide du CNRS (ATP N^o A655 1799) et de l'INSERM (AT N^o 74142036)