Nuclear chromatin texture and sensitivity to DNase I in human leukaemic CEM cells incubated with nanomolar okadaic acid

It is now known that the analysis of chromatin texture can be used in oncology as a sensitive detection method, either to define diagnostic classifications or to locate a lesion along a defined trend curve. However, the functional significance of these variations in textural features remains sometimes unclear. Several drugs have been shown to be able to modulate chromatin structure. Among them, the phosphatase inhibitor okadaic acid at low concentration can increase accessibility to DNA in chromatin of carcinoma cells. This paper demonstrates that short exposures (0–3h) to a 10-nM dose of okadaic acid induced an increased sensitivity to DNase I digestion in human CEM leukaemic cell nuclei and that this sensitization was associated to variations of nuclear texture characteristics, as evaluated by image cytometry. CEM cells treated with okadaic acid for 0–3h displayed changes in chromatin supraorganization with a more homogeneous and fine chromatin texture, as compared to control cells. This suggests that the appearance of an open configuration of chromatin structure as evaluated by biochemical methods corresponds to a more decondensed texture of nuclei measured by image cytometry. Longer exposures (6–24h) of CEM cells to 10nM okadaic acid lead to apoptosis. As reported previously for camptothecin-treated HL60 cells, okadaic acid-treated CEM cells display biphasic nuclear chromatin texture changes, i.e. a decondensation phase followed by the appearance of typical apoptotic cells with a smaller nuclear area and a highly condensed chromatin. Finally, using the multidrug-resistant CEM-VLB cell line, it was confirmed that these multidrug-resistant cells also display cross-resistance to okadaic acid, as this compound was unable to induce either increased DNase I sensitivity, apoptosis, or altered nuclear texture in this particular cell line.     

Nuclear DNA content and chromatin pattern of rat rhabdomyosarcoma cell sublines with different metastatic potentials.

There is a constant need of features able to characterize potentially metastatic cells among the heterogeneous cell subpopulations which constitute a tumor. Image cytometry of metastatic tumor cells give rise to variable results, partly because of a heterogeneous origin of cells, or potential drug effects. The aim of this work was to characterize nuclear changes observed in metastatic cell clones issued in vitro from the same parental cell population The nuclear phenotypes of 6 cell sublines isolated from a rat rhabdomyosarcoma cell line and differing in their metastatic ability were evaluated by image cytometry on Feulgen-stained preparations. Densitometric [5], geometric [3] and textural [9] features were computed from each nuclear image. For each cell subline, a metastatic score, ranging from 0 to 10, was calculated on the basis of in vivo invasivity data, by measuring the number of pulmonary metastases observed after s.c. graft of tumor cells in rats. Data obtained were compared to karyotype, growth characteristics, and oncogene expressions of cell lines. The nuclear DNA content, the chromosome numbers, the cell sublines doubling times, and the distribution of cells within the cell cycle appear unrelated with this score. On the contrary, increase in metastatic ability is accompanied by changes in chromatin pattern as assessed by textural features. Progressive increase in chromatin condensation can be observed in cell sublines with increasing metastatic score. These results were confirmed by an unsupervised multivariate partitioning of rhabdomyosarcoma cells which identified two separate subsets whose distributions within the analyzed cell lines correlate with their metastatic ability. These data suggest that, in rat rhabdomyosarcoma cell sublines, metastatic ability could be associated with nuclear morphological changes at the level of chromatin texture.     

Nuclear texture parameters as discriminant factors in cell cycle and drug sensitivity studies

The authors have recently shown that cell cycle characteristics of in situ cell populations can be determined using the SAMBA 200 cell image processor by computing 15 densitometric and texture parameters on each Feulgen-stained nucleus and multiparametric analysis of data. The present paper displays the importance of chromatin pattern assessment and detection of conformational changes in DNA structure, based on nine nuclear texture parameters measured from the grey level cooccurrence and the run-length section matrices. Reference files were constructed by merging respective reference files (G0/G1, S, G2 and M) of MDA AG and MCF-7, two mammary epithelial cell lines presenting different morphological aspects and hormone responses, these files were found to be valid in the reclassification of any mammary epithelial cell in culture with a diploid or near diploid pattern. Moreover, the authors demonstrate that chromatin texture changes, following direct interaction of chemotherapeutic drugs with DNA, may be assessed owing to nuclear texture parameters. Consecutive to daunomycin addition (0.5 microgram/ml) and concomitant to the appearance of nuclear morphological alterations in MDA AG sensitive cells as viewed by microscopic observation, discriminant factorial analysis showed progressively increasing erroneous reclassification from 15 to 72 h of treatment. These experimental results were exploited with a kinetic mathematical model to quantify the daunomycin blocking effect: 20% in S phase and 80% in G2 phase. Interestingly, no textural change was observed on MDA A1 anthracycline resistant cells, indicating that these texture parameters may permit distinction of drug sensitive cells. This methodology 1) can be applied to test in vitro resistance-reversal molecules, 2) may be extended to other therapeutic agents giving rise to conformational changes in DNA structure, and 3) can be applied to cytopunctions or imprints of tumor biopsies with diploid-like DNA content to follow evolution of drug sensitivity or resistance during course of therapy.     

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL－60 cells

INTRODUCTIIONThe nuclear matrix is an essential component ofthe nucleus which is important for the nuclear structural integrity and specific genomic functions[1, 2].Several articles have reported that the nuclear matrix, as a higher order framework structures, mightbe disassembled du-ring the apoptotic process[3-5].Accordingly3 nuclear lamins A/C or B have beenfound to decrease in apoptotic thymocytes[6], Tcells[7], and carcinoma cell line[8, 9]. The nucleolar protein B23, an obscure ma…     

Chromatin Texture Analysis in Living Cells

In recent years, there has been an increasing interest in applications of fluorescence measurements to studies on many physiological mechanisms in living cells. However, few studies have taken advantage of DNA quantification by fluorometry for dynamic assessment of chromatin organization. This type of approach involves both optimal conditions for DNA staining and the use of image cytometry. In this context, this report describes the application of an internal grey-level segmentation method for the assessment of real time modifications of chromatin organization in living cells. These developments are based on a specific, stoichiometric method for nuclear DNA content measurement. Preliminary data obtained from Hela cells suggests the possibility of following variations of nuclear texture (heterogeneity, granularity, condensation, radial distribution) related to the cell cycle progression of cells that are maintained alive.     

NuMA interaction with chromatin is vital for proper chromosome decondensation at the mitotic exit

NuMA is an abundant long coiled-coil protein that plays a prominent role in spindle organization during mitosis. In interphase, NuMA is localized to the nucleus and hypothesized to control gene expression and chromatin organization. However, because of the prominent mitotic phenotype upon NuMA loss, its precise function in the interphase nucleus remains elusive. Here, we report that NuMA is associated with chromatin in interphase and prophase but released upon nuclear envelope breakdown (NEBD) by the action of Cdk1. We uncover that NuMA directly interacts with DNA via evolutionarily conserved sequences in its C-terminus. Notably, the expression of the DNA-binding–deficient mutant of NuMA affects chromatin decondensation at the mitotic exit, and nuclear shape in interphase. We show that the nuclear shape defects observed upon mutant NuMA expression are due to its potential to polymerize into higher-order fibrillar structures. Overall, this work establishes the spindle-independent function of NuMA in choreographing proper chromatin decompaction and nuclear shape by directly associating with the DNA.     

Altered DNA topoisomerase II in multidrug resistance

The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo II gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected. We propose that MDR associated with alterations in topo II may have clinical consequences, and our current efforts involve exploiting these biochemical and molecular observations in the development of probes that may be useful to identify such drug resistant cells in the tumors of patients.     

S phase, an evolutionary chromatin condensation state from G1 to G2, in a breast epithelial cell line.

In order to better understand the changes in DNA organization during the cell cycle, we quantified the chromatin texture of breast epithelial cells and followed its evolution through a cell cycle. The diversity of quiescent cell states led us to limit this study to proliferating cell phases, and to choose a cell line with no G0 cells, the MDA AG cell line. We recently developed a methodology for characterizing in situ the cell cycle of breast epithelial cell lines using a cell image processor. This method is based on 15 densitometric and texture parameters computed on individual Feulgen-stained nuclei and on multiparametric analysis of the resulting data. Chromatin pattern assessment is based on nine texture parameters measured from grey-level co-occurrence and run-length section matrices. In the present study, texture parameter computation showed gradual and progressive modifications of nuclear texture. While discrimination of G1, G2 and M phases was possible, we could not discriminate G1 from S and S from G2. The chromatin pattern (defined by these nine parameters) in the G1 and early S phases, on the one hand, and in the late S and G2 phases, on the other hand, were similar. The parameter values of cells in the S phase progressively increased from G1 to G2. Two interphase chromatin condensation states were distinguished in these breast cells: a base state characteristic of a prereplicative stage and a very granular state characteristic of a postreplicative stage. We hypothesized that S cells are a blend of these two states, the evolution of a non-duplicated state toward a duplicated one.     

Correlation between chromatin morphology as derived by digital image analysis and autoradiographic labeling pattern

In order to interpret the Feulgen-dependent chromatin morphology on a functional basis, we performed model experiments in which labeling with 14C-thymidine and 14C-uridine was used as a functional parameter. Using a relocation facility, information on either DNA or RNA, labeling intensity of a cell was added to the parameters of image analysis by measuring the same cell by scanning photometry after Feulgen staining. The Feulgen-stained nuclei were interactively sampled and automatically segmented. Most of the textural information was gained from a flat texture image obtained by subtracting the original image from a median-filtered image. In addition to the autoradiographic features, visually recognizable differences in nuclear morphology, such as the number of nucleoli and the level of condensed (inactive) and diffuse (active) regions of the chromatin, were also correlated with textural parameters. Using the supervised cluster analysis method, an attempt was made to establish a correlation between visual nuclear morphology and autoradiographic labeling intensity that improved the functional understanding of the Feulgen features. Our results further clarify the supramolecular chromatin structure and its dynamics during specific transitions in the cell cycle, namely the G0-G1, G1-S and S-G2 transitions; this information may become useful in diagnostic procedures.     

Epigenetically induced changes in nuclear textural patterns and gelatinase expression in human fibrosarcoma cells

Objective

Chromatin texture patterns of tumour cell nuclei can serve as cancer biomarkers, either to define diagnostic classifications or to obtain relevant prognostic information, in a large number of human tumours. Epigenetic mechanisms, mainly DNA methylation and histone post‐translational modification, have been shown to influence chromatin packing states, and therefore nuclear texture. The aim of this study was to analyse effects of these two mechanisms on chromatin texture, and also on correlation with gelatinase expression, in human fibrosarcoma tumour cells.

Materials and methods

We investigated effects of DNA hypomethylating agent 5‐aza‐2′‐deoxycytidine (5‐azadC) and histone deacetylase inhibitor trichostatin A (TSA) on nuclear textural characteristics of human HT1080 fibrosarcoma cells, evaluated by image cytometry, and expression of gelatinases MMP‐2 and MMP‐9, two metalloproteinases implicated in cancer progression and metastasis.

Results

5‐azadC induced significant variation in chromatin higher order organization, particularly chromatin decondensation, associated with reduction in global DNA methylation, concomitantly with increase in MMP‐9, and to a lesser extent, MMP‐2 expression. TSA alone did not have any effect on HT1080 cells, but exhibited differential activity when added to cells treated with 5‐azadC. When treated with both drugs, nuclei had higher texture abnormalities. In this setting, reduction in MMP‐9 expression was observed, whereas MMP‐2 expression remained unaffected.

Conclusions

These data show that hypomethylating drug 5‐azadC and histone deacetylase inhibitor TSA were able to induce modulation of higher order chromatin organization and gelatinase expression in human HT1080 fibrosarcoma cells.

     

Analysis of DNA content in multidrug-resistant cells by image and flow cytometry

Abstract. Nuclear DNA content was assessed in multidrug-resistant (MDR) cells by image and flow cytometry. Two human MDR cell lines (K562-Dox and CEM-VLB) obtained by in vitro drug selection and overexpressing mdr1 gene were compared to their respective sensitive counterparts (K562 and CCRF-CEM) and to the MDR hamster LR73-R cell line obtained by transfection of mouse mdr1 cDNA. Both cell lines obtained by selection displayed a decreased DNA content, as measured by image cytometry after Feulgen staining, or by flow cytometry after staining with propidium iodide, ethidium bromide, or Hoechst 33342. This decrease was not accompanied by changes in cell cycle phase distribution of cells. Moreover, image cytometry of cells stained after various hydrolysis times in 5 M HCl indicated that MDR cells displayed the same hydrolysis kinetics and sensitivity as drug-sensitive cells with a well-preserved stoichiometry of the Feulgen reaction. LR73-R cells transfected with mdr1 cDNA exhibited only a very limited change in propidium iodide staining as compared with sensitive LR73 cells, suggesting that mdr1 gene overexpression alone could not account for the alterations in DNA content observed in the selected MDR cells.     

Apoptotic cleavage of NuMA at the C-terminal end is related to nuclear disruption and death amplification

NuMA is a nuclear matrix protein in interphase and distributes to the spindle poles during mitosis. While the essential function of NuMA for mitotic spindle assembly is well established, a structural role of NuMA in interphase nucleus has also been proposed. Several observations suggest that the apoptotic degradation of NuMA may relate to chromatin condensation and micronucleation. Here we demonstrate that four apoptotic cleavage sites are clustered at a junction between the globular tail and the central coiled-coil domains of NuMA. Cleavage of a caspase-6-sensitive site at D¹⁷⁰⁵ produced the R-form, a major tail-less product of NuMA during apoptosis. The other two cleavage sites were defined at D¹⁷²⁶ and D¹⁷⁴⁷ that were catalyzed, respectively, by caspase-3 and an unknown aspartase. A NuMA deletion mutant missing the entire cleavage region of residues 1701–1828 resisted degradation and protected cells from nuclear disruption upon apoptotic attack. Under such conditions, cytochrome c was released from mitochondria, but the subsequent apoptotic events such as caspase-3 activation, poly(ADP-ribose) polymerase degradation, and DNA fragmentation were attenuated. Conversely, the tail-less NuMA alone, a mutant mimicking the R-form, induced chromatin condensation and activated the death machinery. It supports that intact NuMA is a structural element in maintaining nuclear integrity.     

Changes of chromatin organization induced by phospholipids

Isolated nuclei represent a suitable model for studying the influence of exogenous phospholipids, normally found as minor chromatin components, on the nuclear structure, which, in turn, could be related to the observed modifications of DNA and RNA synthesis. The morphological modifications induced on chromatin RNP granules and nuclear matrix have been analyzed both with conventional thin sectioning and with an original method based on image analysis of freeze-fractured and replicated nuclear samples. The results obtained support the hypothesis that anionic phospholipids, by removing histone H1, induce a transition of the chromatin from solenoid to nucleosome conformation and favour the RNA polymerizing activity which results in an increased release of RNP particles, while neutral phospholipids, probably affecting the matrix structure, partly impare the RNP maturation and transport, with consequent increase of chromatin condensation.     

Caspase-3 is required in the apoptotic disintegration of the nuclear matrix

Apoptotic breakdown of cellular structures is largely mediated by caspases. One target of degradation is a proteinaceous framework of the nucleus termed the nuclear matrix. We compared the apoptotic changes of the nuclear matrix in staurosporine-treated caspase-3-deficient MCF-7 cells transfected with intact CASP-3 gene (MCF-7c3) or an empty vector (MCF-7v) as a control. Nuclear Mitotic Apparatus protein (NuMA), lamin A/C and lamin B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. In both cell lines, staurosporine induced rapid cytoplasmic shrinkage and partial chromatin condensation. MCF-7c3 cells formed apoptotic bodies, whereas MCF-7v cells did not. NuMA and lamins were actively cleaved in MCF-7c3 cells following caspase-3 activation, but only minimal or no cleavage was detected in MCF-7v cells. Interestingly, lamin B but not lamin A/C was relocated into cytoplasmic granules in apoptotic MCF-7v cells. Pancaspase inhibitor, z-VAD-fmk, prevented the apoptotic changes, while caspase-3 inhibitor, z-DEVD-fmk, induced lamin B granules in both cell lines. These results show that caspase-3 is involved in the cleavage of NuMA and lamins either directly or by activating other proteases. This may be essential for disintegration of the nuclear structure during apoptosis.     

Silencing of Nuclear Mitotic Apparatus protein (NuMA) accelerates the apoptotic disintegration of the nucleus

One main feature of apoptosis is the sequential degradation of the nuclear structure, including the fragmentation of chromatin and caspase-mediated cleavage of various nuclear proteins. Among these proteins is the Nuclear Mitotic Apparatus protein (NuMA) which plays a specific role in the organization of the mitotic spindle. The exact function of NuMA in the interphase nucleus is unknown, but a number of reports have suggested that it may play a role in chromatin organization and/or gene expression. Here we show that upon cleavage in apoptotic cells, the N-terminal cleavage fragment of NuMA is solubilized while the C-terminal fragment remains associated with the condensed chromatin. Using pancaspase inhibitor z-VAD-fmk and caspase-3 deficient MCF-7 cells, we further show that the solubilization is dependent on caspase-mediated cleavage of NuMA. Finally, the silencing of NuMA by RNAi accelerated nuclear breakdown in apoptotic MCF-7 cells. These results suggest that NuMA may provide structural support in the interphase nucleus by contributing to the organization of chromatin.     

植物细胞中NuMA类似蛋白的分布及其在细胞周期中的变化

免疫荧光染色结果说明植物细胞核内含有与抗动物ＮｕＭＡ多抗呈阳性交叉反应的多肽。选择性抽提并结合免疫荧光染色结果说明这种多肽位于核基质纤维蛋白网络上。免疫印迹反应显示胡萝卜（ＤａｕｃｕｓｃａｒｏｔａＬ．）悬浮培养细胞核基质蛋白与抗动物ＮｕＭＡ蛋白多抗的阳性反应条带为７４ｋＤ和７６ｋＤ。有丝分裂各期免疫荧光染色的结果表明植物细胞中的ＮｕＭＡ类似蛋白在有丝分裂过程中呈现有规律的变化。结合选择性抽提的有丝分裂各期的免疫荧光染色的结果表明核基质在此过程中也发生明显变化。应用选择性抽提并结合ＤＧＤ包埋去包埋电镜技术对植物细胞间期及有丝分裂期核基质的形态结构进行了观察。结果显示胡萝卜悬浮培养细胞间期核内存在一个非染色质性的纤维蛋白网络体系,而在正处于分裂的细胞中则未观察到。以上结果说明ＮｕＭＡ类似蛋白是核基质的组分之一并与有丝分裂密切相关。     

Nuclear image analysis of immunohistochemically stained cells in breast carcinomas

Hitherto, the relationship between malignancy-associated morphological features in single tumour cells and the expression of markers indicating functional properties of these cells remained widely unknown. This study was aimed at describing differences in the size, shape and chromatin structure between tumour cells with different marker expression for progesterone receptors (PgR) and p53. Two series of breast cancers, consisting of 50 PgR-positive, and 39 p53-negative and 49 p53-positive mammary carcinomas, were investigated. The immunohistochemical staining was performed on paraffin sections using 3-amino-9-ethylcarbazole as the chromogenic substrate. By means of a cytometry workstation equipped with a computer-controlled motorised scanning stage, about 500 positive and negative tumour cells in each case were localised in the microscope and categorised by a scoring system for their staining intensity. After destaining, the tissue sections were Feulgen-stained. Then, all the tumour cells were relocated automatically and analysed by high resolution image cytometry. Among the numerous size, shape, and texture features used in the system, several variables of the nuclear contour and chromatin structure were found to be significantly different between the positive and negative tumour cell populations. Nuclei without PgR had more malignancy-associated morphological features than PgR-positive cells. Whereas p53-negative nuclei had a higher degree of regularity, their positive counterparts exhibited higher DNA ploidy values.