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1.
Food Chem ; 218: 356-364, 2017 Mar 01.
Article in English | MEDLINE | ID: mdl-27719921

ABSTRACT

Vitexin-2-O-xyloside (XVX) from Beta vulgaris var. cicla L. (BVc) seeds, betaxanthin (R1) and betacyanin (R2) fractions from Beta vulgaris var. rubra L. (BVr) roots were combined and tested for cytotoxicity in CaCo-2 colon cancer cells. XVX was the most cytotoxic molecule, but the combination of XVX with R1 and R2 significantly prolonged its cytotoxicity. Cytotoxicity was mediated by the intrinsic apoptotic pathway, as shown by an increase in Bcl2-like protein 4, cleaved Poly ADP-Ribosyl Polymerase 1 and cleaved Caspase 3 levels with a parallel decrease in anti-apoptotic protein B-cell leukemia/lymphoma 2 levels. R1 and R2, used alone or in combination, reduced oxidative stress triggered by H2O2 in CaCo-2 cells. Betalains dampened cyclooxygenase-2 and interleukin-8 mRNA expression after lipopolysaccharide induction in CaCo-2, showing an anti-inflammatory action. Our results support the use of a cocktail of R1, R2 and XVX as a chemopreventive tool against colon cancer.


Subject(s)
Beta vulgaris/chemistry , Betalains/pharmacology , Flavonoids/pharmacology , Glycosides/pharmacology , Anti-Inflammatory Agents/pharmacology , Apoptosis/drug effects , B-Lymphocytes/metabolism , Caco-2 Cells , Caspase 3/genetics , Caspase 3/metabolism , Chemoprevention , Colonic Neoplasms/drug therapy , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Humans , Hydrogen Peroxide , Interleukin-8/genetics , Interleukin-8/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oxidative Stress/drug effects , Plant Extracts/pharmacology , Plant Roots/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism
2.
J Anal Toxicol ; 38(2): 80-5, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24396052

ABSTRACT

A simple, fast and cost-effective liquid chromatographic/tandem mass spectrometry (LC-MS-MS) method for the quantitative determination of flunixin (FLU) in bovine muscle was developed and validated. The sample preparation procedure involved an extraction with acetonitrile, followed by evaporation and reconstitution. Chromatographic separation was achieved on a reverse-phase column under programmed conditions. FLU detection was performed with positive electrospray ionization in selected reaction monitoringmode, monitoring one precursor and two products ions. For quantification purposes, FLU-d3 was used as an internal standard. The matrix effect on the analysis of FLU in bovine muscle was evaluated by comparison between calibration curves prepared with standard solution and in blank matrix extracts. The equivalent responses obtained confirmed the absence of signal suppression or/and enhancement. The method was extensively validated according to the parameters requested by European Commission Decision 2002/657/EC in terms of specificity, limit of detection, linearity, trueness, precision, decision limit (CCα) and detection capability (CCß). FLU stability was also investigated in matrix and in sample extracts at different times and storage conditions.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/analysis , Chromatography, Liquid , Clonixin/analogs & derivatives , Muscle, Skeletal/chemistry , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Animals , Calibration , Cattle , Chromatography, Liquid/standards , Clonixin/analysis , Drug Stability , Limit of Detection , Reference Standards , Reproducibility of Results , Spectrometry, Mass, Electrospray Ionization/standards , Tandem Mass Spectrometry/standards , Time Factors
3.
Eur J Pharm Sci ; 47(4): 729-38, 2012 Nov 20.
Article in English | MEDLINE | ID: mdl-22954722

ABSTRACT

Galloflavin (GF), a recently identified lactate dehydrogenase inhibitor, hinders the proliferation of cancer cells by blocking glycolysis and ATP production. The aim of the present experiments was to study the effect of this compound on breast cancer cell lines reproducing different pathological subtypes of this tumor: MCF-7 (the well differentiated form), MDA-MB-231 (the aggressive triple negative tumor) and MCF-Tam (a sub-line of MCF-7 with acquired tamoxifen resistance). We observed marked differences in the energetic metabolism of these cell lines. Compared to MCF-7 cells, both MDA-MB-231 and MCF-Tam cells exhibited higher LDH levels and glucose uptake and showed lower capacity of oxygen consumption. In spite of these differences, GF exerted similar growth inhibitory effects. This result was explained by the finding of a constitutively activated stress response in MDA-MB-231 and MCF-Tam cells, which reproduce the poor prognosis tumor forms. As a further proof, different signaling pathways were found to be involved in the antiproliferative action of GF. In MCF-7 cells we observed a down regulation of the ERα-mediated signaling needed for cell survival. On the contrary, in MCF-Tam and MDA-MB-231 cells growth inhibition appeared to be contributed by an oxidative stress condition. The prevalent mechanism of cell death was found to be apoptosis induction. Because of the clinical relevance of breast cancer forms having the triple negative and/or chemoresistant phenotype, our results showing comparable effects of GF even on aggressively growing cells encourage further studies to verify the potential of this compound in improving the chemotherapy of breast cancer.


Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/enzymology , Cell Death/drug effects , Isocoumarins/pharmacology , L-Lactate Dehydrogenase/antagonists & inhibitors , Apoptosis/drug effects , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Energy Metabolism/drug effects , Female , Glucose/metabolism , Glycolysis/drug effects , Humans , Lymphocytes/drug effects , Lymphocytes/metabolism , MCF-7 Cells , Oxidative Stress/drug effects , Oxygen Consumption/drug effects , Signal Transduction/drug effects
4.
Phytomedicine ; 17(5): 356-62, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20149610

ABSTRACT

We investigated the anticancer effect of EGCG treatment on a breast carcinoma cell line resistant to tamoxifen (MCF-7Tam cells). As there are no reports about the molecular mechanisms implicated in EGCG treatment of tamoxifen resistant breast carcinoma cells, we studied the effects of EGCG treatment on three plasma membrane proteins that are involved in the mechanism of drug-resistance: Multidrug Resistance Protein (MRP1), P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). EGCG treatment (10-100 microg/ml for 24-72 hours) caused cell growth inhibition and dose-dependent apoptosis: after 100 microg/ml EGCG treatment for 24 hours, Bax expression increased and Bcl2 expression decreased (p<0.05). Coherently, Annexin V-FITC apoptosis assay detected a significant increase in labelled cells (p<0.05). EGCG did not affect MRP1: in contrast, 100 microg/ml EGCG administration caused P-gp decrease to 53% of control cells (p<0.001) and this effect was not due to downregulation of P-gp gene expression. EGCG induced P-gp decrease even when MG132, a strong proteasome inhibitor, was given together with EGCG to MCF-7Tam cells. EGCG treatment also inhibited BCRP activity: mRNA transcription and protein level did not change after treatment, but mitoxantrone test demonstrated a strong inhibition of BCRP activity (p<0.001). In conclusion, the present results showed that EGCG could down-regulate the activity of two molecules that play a key role in drug metabolism and transport and that are highly expressed in tamoxifen resistant breast carcinoma cells. The interaction of EGCG and drugs used in the therapy of estrogen sensitive breast carcinoma ought to be subject of studies and the potential use of EGCG in drug-resistant diseases ought to be better considered.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Antineoplastic Agents, Phytogenic/therapeutic use , Breast Neoplasms/drug therapy , Catechin/analogs & derivatives , Neoplasm Proteins/metabolism , Plant Extracts/therapeutic use , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Camellia sinensis/chemistry , Catechin/pharmacology , Catechin/therapeutic use , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Down-Regulation , Drug Resistance, Neoplasm/drug effects , Gene Expression , Humans , Leupeptins/pharmacology , Mitoxantrone/pharmacology , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Neoplasm Proteins/genetics , Plant Extracts/pharmacology , Protease Inhibitors/pharmacology , Protease Inhibitors/therapeutic use , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/metabolism , Tamoxifen , bcl-2-Associated X Protein/metabolism
5.
J Clin Pathol ; 58(10): 1046-50, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16189149

ABSTRACT

AIMS: To investigate SOCS-2 (suppressor of cytokine signalling 2) protein expression in breast carcinoma samples in relation to biopathological parameters and survival. METHODS: A polyclonal antibody against SOCS-2 was used to study 50 archival breast carcinoma samples, collected from 1993 to 1995. The presence of SOCS-2 protein was investigated in relation to clinical and biological parameters used in breast cancer pathology. Fluorescence in situ hybridisation (FISH) was used to study whether SOCS-2 expression was related to SOCS-2 gene copy number. RESULTS: SOCS-2 protein was expressed in 34 of 50 breast carcinoma samples and was positively associated with low grade, low nuclear grade, and p27 protein. SOCS-2 expression was inversely related to Ki-67, cyclin A, retinoblastoma protein (pRb), and the epidermal growth factor receptor (EGFR). No relation with overall survival was demonstrated. SOCS-2 amplification was found in three samples. No relation between the number of FISH signals and SOCS-2 expression was found. CONCLUSIONS: The significant correlation seen between SOCS-2 expression, grade, nuclear grade, p27, Ki-67, cyclin A, pRb, and EGFR labelling strongly supports the hypothesis that SOCS-2 loss might be related to cell proliferation and tumour growth in breast carcinoma. Gene copy number changes did not seem to play a role in SOCS-2 regulation and expression; other mechanisms might be involved and deserve further study.


Subject(s)
Breast Neoplasms/metabolism , DNA-Binding Proteins/metabolism , Neoplasm Proteins/metabolism , Repressor Proteins/metabolism , Trans-Activators/metabolism , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/metabolism , Breast Neoplasms/pathology , Cell Cycle Proteins/metabolism , Cell Proliferation , Cyclin A/metabolism , Cyclin-Dependent Kinase Inhibitor p27 , DNA-Binding Proteins/genetics , ErbB Receptors/metabolism , Female , Humans , In Situ Hybridization, Fluorescence , Ki-67 Antigen/metabolism , Middle Aged , Neoplasm Proteins/genetics , Repressor Proteins/genetics , Retinoblastoma Protein/metabolism , Suppressor of Cytokine Signaling Proteins , Survival Rate , Trans-Activators/genetics , Tumor Suppressor Proteins/metabolism
6.
Cytometry ; 46(1): 50-6, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11241507

ABSTRACT

We investigated the relationship between DNA ploidy and alterations in chromosomes 1, 8, 12, 16, 17, and 18 in 63 breast carcinoma samples by static cytofluorometry and fluorescence in situ hybridization. Thirty specimens were diploid and 33 were aneuploid. In aneuploid samples, the DNA index value ranged from 1.3 to 3.1, with a main peak near tetraploid values. Diploid clones were present in 21 of 33 aneuploid specimens. Fluorescence in situ hybridization analysis showed a heterogeneous degree of alterations in diploid specimens: one sample was normal, 16 samples had one to three chromosome alterations involving mostly chromosomes 1, 16, and 17, and 13 samples an even higher degree of alterations. The 33 aneuploid specimens showed a very high number of signals (four, five, or more). All the investigated chromosomes were affected in 23 of 33 specimens. Alterations in chromosomes 1 and 17 were detected to a similar percentage in diploid and aneuploid samples, whereas chromosome 16 monosomy was more frequent in diploid samples. Overrepresentation of chromosomes 8, 12, 16, and 18 was significantly higher in aneuploid than in diploid samples. Based on these results, we suggest that diploid and aneuploid breast carcinomas are genetically related. Chromosome 1 and 17 alterations and chromosome 16 monosomy are early changes. Allelic and chromosomal accumulations occur during progression of breast carcinoma by different mechanisms. The high clone heterogeneity found in 17 of 33 aneuploid samples could not be completely explained by endoreduplication and led to the suggestion that chromosomal instability concurs with aneuploidy development. This different evolutionary pathway might be clinically relevant because clone heterogeneity might cause metastasis development and resistance to therapy.


Subject(s)
Breast Neoplasms/genetics , Carcinoma/genetics , Chromosome Aberrations , Aneuploidy , Chromosomes, Human, Pair 1 , Chromosomes, Human, Pair 12 , Chromosomes, Human, Pair 16 , Chromosomes, Human, Pair 17 , Chromosomes, Human, Pair 18 , Chromosomes, Human, Pair 8 , Diploidy , Female , Flow Cytometry , Humans , In Situ Hybridization, Fluorescence
7.
Int J Cancer ; 84(3): 273-7, 1999 Jun 21.
Article in English | MEDLINE | ID: mdl-10371346

ABSTRACT

We investigated c-erbB-2 oncogene amplification and over-expression in 79 invasive breast carcinoma samples using fluorescence in situ hybridization (FISH) and immunohistochemistry, with the aim of studying relationships between neoplasms over-expressing c-erbB-2 with or without amplification and bio-pathological parameters used in clinical breast cancer. Nineteen samples showed amplification, and all of these were positive by immuno-histochemistry. Moderate or intense immunostaining was present in a further 22 samples without c-erbB-2 amplification and was not related to any increased number of c-erbB-2 signals: 15 samples exhibited chromosome 17 polysomy, 3 monosomy and 4 no FISH abnormalities. Thirty-eight samples were immunonegative: 18 exhibited chromosome 17 polysomy, 9 monosomy and 11 no alterations. Samples having c-erbB-2 over-expression associated with amplification showed DNA aneuploidy and hormonal receptor loss to a greater extent than those expressing c-erbB-2 without amplification or immunonegative samples (chi2 test, p = 0.007, 0.008 and 0.008, respectively). The proliferation rate, detected by Ag-NOR staining, was highest in amplified samples (Kruskal Wallis test, p = 0.009). Our results indicate that tumours showing both c-erbB-2 over-expression and amplification exhibit more aggressive biological characteristics than those with only over-expression or immunonegative tumours. Since both c-erbB-2 amplification and over-expression have been related to negative responses to chemotherapy and poor prognosis, these differences might have clinical implications. The combination of FISH and immuno-histochemistry may be helpful to achieve this aim.


Subject(s)
Breast Neoplasms/chemistry , Gene Amplification , Genes, erbB-2 , Receptor, ErbB-2/analysis , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Female , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence
8.
Eur J Cancer ; 34(5): 724-30, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9713281

ABSTRACT

A new human cancer cell line was established from a metastatic lesion of a small cell lung carcinoma (SCLC-R1) and maintained in continuous culture with a doubling time of 62 h. The SCLC-R1 line, whose ultrastructural features are presented, showed a diploid DNA content, a translocation involving chromosome 16 [t(16;?)(q24;?)] and noticeable deletions in the FHIT (fragile histidine triad) region in the short arm of chromosome 3 [del(3)(p14)] and in the telomeric region of the short arm of chromosome 12 [del(12)(p13)]. The involvement of 12p in metastatic small cell lung cancer is reported here for the first time. No amplification or rearrangements were evident in the c-myc, L-myc, N-myc, int-2, c-erbB-2, H-ras, K-ras, c-mos, and hst-1 genes by Southern blot analysis. Wild-type p53, RB, K-ras and H-ras genes were evident by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. The neuron specific enolase (NSE) level was much higher in the cell line's cytosol than in the patient's serum and the cell line also had high expression of chromogranin A and cytokeratin 19. SCLC-R1 cells were sensitive to cisplatin, carboplatin and doxorubicin. The clinical history of the patient from whom the cell line was derived is reported. The characteristics of this new cell line indicate it to be a useful experimental model to investigate lung cancer biology and anticancer drug response.


Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Small Cell/genetics , Chromosome Aberrations , Lung Neoplasms/genetics , Tumor Cells, Cultured/drug effects , Aged , Biomarkers, Tumor/metabolism , Carcinoma, Small Cell/drug therapy , Carcinoma, Small Cell/metabolism , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Diploidy , Gene Deletion , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Male , Proto-Oncogene Proteins/metabolism , Translocation, Genetic , Tumor Cells, Cultured/pathology
9.
Int J Cancer ; 69(5): 381-5, 1996 Oct 21.
Article in English | MEDLINE | ID: mdl-8900371

ABSTRACT

Chromosome 1 abnormalities with loss of 1p36 have been investigated in 95 breast-cancer samples by means of a dual-target fluorescence in-situ hybridization (FISH) technique using the pUC 1.77 and p1-79 probes, specific for the 1q12 and 1p36 regions, respectively. Abnormalities for one or both probes were detected in 83/95 samples. Relative 1p36 under-representation was found in 79/95. The clinical relevance of these alterations was studied by comparing the FISH results with several parameters currently used in breast-cancer pathology. Distinct patterns of chromosome 1 abnormalities were found among the histologic types of breast carcinoma. Lobular or mucinous samples showed few or no alterations, whereas most ductal samples had high chromosome 1 polysomy with under-representation of 1p36. In ductal carcinomas, chromosome 1 alterations increased with histologic grade, DNA aneuploidy, loss of bcl-2 and high c-erb B-2 expression. These associations were found to be statistically significant. No correlation between chromosome 1 alterations and nuclear grade, age, size, lymph-node involvement, hormonal receptor presence, proliferation activity or p53 protein expression was detected. These results indicate the utility of this FISH technique for a better definition of the biological characteristics of ductal carcinomas.


Subject(s)
Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/pathology , Chromosomes, Human, Pair 1/genetics , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Receptor, ErbB-2/biosynthesis , Adult , Aged , Aneuploidy , Breast Neoplasms/chemistry , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/chemistry , Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/metabolism , Cell Division , Chromosome Deletion , Female , Humans , In Situ Hybridization, Fluorescence , Lymphocytes/chemistry , Middle Aged , Receptors, Estrogen/analysis , Receptors, Progesterone/analysis , Tumor Suppressor Protein p53/biosynthesis
10.
Clin Mol Pathol ; 49(2): M98-M103, 1996 Apr.
Article in English | MEDLINE | ID: mdl-16696058

ABSTRACT

Aims-To investigate whether deletion of the 1p36 region of chromosome 1 is independent of DNA ploidy in breast cancer cells.Methods-Preparations of nuclei from 64 fresh primary breast tumours were studied using dual target fluorescence in situ hybridisation (FISH) combining probes specific for the 1q12 (pUC 1.77) and 1p36 (1p-79) regions of chromosome 1. Signals were counted in 100-300 nuclei and the percentage of cells showing fewer p1-79 than pUC 1.77 signals was measured in each sample. DNA ploidy was investigated by cytofluorimetry in 55 tumour samples.Results-Chromosome 1 aberrations were detected in 56 samples. There were fewer p1-79 than pUC 1.77 signals in 53 samples. The 1p36 region was deleted in 11 samples in which a single p1-79 signal was detected; seven of these samples were diploid. Abnormalities were found in 17/24 diploid and 30/31 aneuploid tumours.Conclusions-Chromosome 1 aberrations, including deletion of the 1p36 region, were observed in diploid breast tumours. Deletion of the 1p36 region may be an early event in tumorigenesis. Given the frequency and importance of chromosome 1 aberrations in the biological behaviour of breast tumours, FISH, used in conjunction with cytofluorimetry, may be helpful for determining prognosis in patients with diploid tumours.

11.
Exp Cell Res ; 211(1): 36-41, 1994 Mar.
Article in English | MEDLINE | ID: mdl-7510250

ABSTRACT

The relationship between topoisomerase II activity and ribosomal RNA synthesis was investigated using the antitumoral drug VM26, a specific inhibitor of topoisomerase II. For this purpose TG cells, a human tumor cell line, were cultured in the presence of 2.5 microM VM26 for 1 and 3 h; VM26 reduced the topoisomerase II activity, measured in whole cell extracts. In the presence of VM26 the [3H]uridine incorporation into ribosomal RNA was decreased; electron microscopy investigation of nucleoli showed a segregation of nucleolar components. Because VM26 stabilizes the cleavable complex and inhibits the resealing reaction, thus causing potential cleavage sites, we have analyzed the double-strand breaks caused by the drug treatment in the tandem repeat ribosomal DNA (rDNA) genes, by indirect labeling with two probes recognizing the 5' portion of ETS (BES) and the 3' portion of 28S (LS6BE) transcribed gene. In VM26-treated cells rDNA is fragmented and a topoisomerase II preferential cleavage site is present, localized at 1.85 kb in 28S region from 3' EcoRI site.


Subject(s)
Cell Nucleolus/physiology , Cell Nucleolus/ultrastructure , DNA Topoisomerases, Type II/metabolism , Topoisomerase II Inhibitors , DNA/analysis , DNA/genetics , DNA Damage , DNA Topoisomerases, Type II/physiology , Humans , Microscopy, Electron , RNA/analysis , RNA/genetics , Teniposide/pharmacology , Tritium , Tumor Cells, Cultured , Uridine/metabolism
12.
J Histochem Cytochem ; 41(6): 829-36, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8315275

ABSTRACT

We studied the distribution of DNA in human circulating lymphocyte nucleoli using three different cytochemical methods for selective visualization of DNA in thin sections: the Feulgen-like osmium-ammine reaction, the NAMA-Ur procedure, and the osmium-ammine staining in glycine buffer, pH 1.5. All three methods indicated the presence of uniformly distributed, highly decondensed DNA filaments forming a large solitary agglomerate in the central part of the nucleolar area, corresponding to the solitary large fibrillar center (FC) as revealed by uranium and lead staining. We also studied the relationship between DNA agglomerates and nucleolar fibrillar components in resting and phytohemagglutinin (PHA)-stimulated lymphocytes by morphometric analysis of the areas occupied by these structures. In resting lymphocytes the mean area of the DNA agglomerates was 0.479 micron 2 +/- 0.161 SD, whereas that of FCs was 0.380 micron 2 +/- 0.149 SD, with a ratio of 1.26. In PHA-stimulated lymphocytes the mean area of the DNA agglomerates was 0.116 micron 2 +/- 0.056 SD, whereas that of the FCs was 0.075 micron 2 +/- 0.032 SD, with a ratio of 1.55. In PHA-stimulated lymphocytes we also measured the area occupied by the FCs plus the closely associated dense fibrillar component (DFC). The mean value of these two fibrillar components was 0.206 micron 2 +/- 0.081 SD. These data demonstrate that decondensed DNA filaments are uniformly distributed in the FCs and that in transcriptionally active nucleoli they are also present in the proximal portion of the DFC surrounding the FCs.


Subject(s)
Cell Nucleolus/metabolism , DNA/metabolism , Cells, Cultured , Humans , Lymphocytes/drug effects , Lymphocytes/metabolism , Phytohemagglutinins/pharmacology
13.
Histochem J ; 24(12): 951-6, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1283387

ABSTRACT

We have studied the relationship between interphase nucleolar organizer region (NOR) distribution and nucleolar size in cancer cells at light-microscopical level. Thirteen cases of formalin-fixed bladder cancer and fifteen cases of methacarn-fixed tumours of different origin were used. Nucleoli of the former cases were stained by Phloxine B and of the latter by Toluidine Blue. Selective visualization of interphase NORs was obtained by carrying out the one-step silver staining reaction for AgNOR proteins (Ploton et al., 1986). The area occupied by Phloxine B- or Toluidine Blue-stained nucleoli and interphase silver-stained NORs was measured by means of an automated image analyser. Both in bladder cancers and in the other tumour lesions nucleolar and interphase AgNOR areas were linearly related (r = 0.95 and r = 0.96, respectively, P < 0.001). The close relationship between the area of nucleoli and that of silver-stained nucleolar structures was maintained even if the silver-staining procedure was prolonged beyond the optimal time length for selective interphase NOR staining. In the latter case, however, single interphase AgNORs were no longer visible within the nucleolar body which was, in fact, homogeneously stained. These data indicate that evaluation of the interphase AgNOR area has the same relevance, in tumour pathology, as whole nucleolar size measurement.


Subject(s)
Cell Nucleolus/ultrastructure , Neoplasms/ultrastructure , Nucleolus Organizer Region/ultrastructure , Eosine I Bluish , Humans , Interphase , Silver , Staining and Labeling , Tolonium Chloride , Urinary Bladder Neoplasms/ultrastructure
14.
Histochemistry ; 98(2): 121-9, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1385369

ABSTRACT

Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37 degrees C of isolated nuclei. If purified nuclei were heated for 45 min at 37 degrees C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-amine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.


Subject(s)
Friend murine leukemia virus , Leukemia, Erythroblastic, Acute/pathology , Nuclear Matrix/ultrastructure , Animals , DNA, Neoplasm/metabolism , Deoxyribonucleases/metabolism , Freeze Fracturing , Histocytochemistry , Mice , Plastic Embedding , RNA, Neoplasm/metabolism , Staining and Labeling , Tumor Cells, Cultured
15.
Biol Cell ; 74(3): 281-6, 1992.
Article in English | MEDLINE | ID: mdl-1320976

ABSTRACT

The effects of camptothecin treatment and topoisomerase I inhibition on ribosomal gene structure and function were investigated in TG cells, a human tumour cell line. 90- and 180-min treatments with 25 microM camptothecin resulted in an increased DNA fragmentation and decreased activity of topoisomerase I in cell extracts. After 180-min treatment, the incorporation of labelled uridine into total cell RNA was reduced to 39% and the ribosomal RNA synthesis to 10%, as compared to values of control cells. At the ultrastructural level, the nucleolar components appeared to be segregated; after selective DNA staining, with osmium-amine complex, a part of the nucleolar chromatin of treated cells showed the presence of thin, extended DNA filaments, superimposable to those present in control cells.


Subject(s)
Camptothecin/pharmacology , Cell Nucleolus/drug effects , DNA, Neoplasm/drug effects , DNA, Ribosomal/drug effects , RNA, Neoplasm/genetics , RNA, Ribosomal/genetics , Topoisomerase I Inhibitors , Cell Nucleolus/ultrastructure , Chromatin/drug effects , Chromatin/ultrastructure , DNA Damage , Depression, Chemical , Genes/drug effects , Humans , RNA, Neoplasm/biosynthesis , RNA, Ribosomal/biosynthesis , Transcription, Genetic/drug effects , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Tumor Cells, Cultured/ultrastructure
16.
J Pathol ; 165(1): 53-9, 1991 Sep.
Article in English | MEDLINE | ID: mdl-1955936

ABSTRACT

The area of silver-stained proteins associated with interphase nucleolar organizer regions (AgNORs) was compared with labelling data obtained by bromodeoxyuridine (BrdU) incorporation and Ki-67 immunostaining in 25 tumours of different origins and two non-neoplastic lesions of the thyroid. Our data demonstrate a highly significant correlation between the mean area occupied by the AgNOR proteins measured by an image processing system and the proliferative indices evaluated by BrdU labelling (r = 0.89, P less than 0.001) and Ki-67 immunostaining (r = 0.86, P less than 0.001). AgNOR protein area measurement is therefore proposed as a simple, inexpensive, and reliable method of evaluating the proliferative activity in routinely processed tumour samples.


Subject(s)
Neoplasms/ultrastructure , Nucleolus Organizer Region/ultrastructure , Breast Neoplasms/ultrastructure , Bromodeoxyuridine/pharmacology , Colonic Neoplasms/ultrastructure , Goiter, Nodular/pathology , Humans , Interphase , Liver Neoplasms/ultrastructure , Mitosis , Nuclear Proteins/analysis , Thyroid Neoplasms/ultrastructure
17.
Chromosoma ; 100(4): 242-50, 1991 May.
Article in English | MEDLINE | ID: mdl-1711437

ABSTRACT

The relationship between the Ag-NOR (silver-stained Nucleolar Organizer Region) proteins and the functional-structural organization of the nucleolar ribosomal chromatin was studied in regenerating and cortisol-stimulated rat hepatocytes. Statistical analysis of Ag-NOR proteins, carried out with an automated image analyzer, indicated that in regenerating rat hepatocytes the quantity of Ag-NOR proteins mainly increased between the 4th and 12th h of regeneration, reaching a level twice that of resting hepatocytes. Also the synthesis of pre-ribosomal RNA (pre-rRNA) was stimulated after the 4th h of regeneration. Cycloheximide administered to rats at a dose of 0.025 mg/100 g body weight (bw) prevented any increase in Ag-NOR proteins but did not hinder the stimulation of pre-rRNA synthesis. In 8 h cortisol-stimulated hepatocytes no significant change in amount of Ag-NOR protein was observed whereas pre-rRNA synthesis was highly increased as in 12 h regenerating hepatocytes. These results indicated that in rat hepatocytes Ag-NOR proteins and stimulation of pre-rRNA synthesis are not related. The relationship between the Ag-NOR proteins and the distribution of the completely extended intranucleolar ribosomal chromatin was also studied in regenerating rat hepatocytes. At 12 h after partial hepatectomy an increased amount of completely extended ribosomal chromatin was observed, contemporaneously with an increased quantity of Ag-NOR proteins. These ribosomal chromatin changes preceded the beginning of DNA synthesis and were prevented by cycloheximide-induced inhibition of protein synthesis.


Subject(s)
Multigene Family , Nuclear Proteins/genetics , Nucleolus Organizer Region/metabolism , RNA, Ribosomal/biosynthesis , Animals , Cell Cycle , DNA/biosynthesis , Liver/cytology , Liver/ultrastructure , Liver Regeneration , Microscopy, Electron , Nucleolus Organizer Region/ultrastructure , Rats , Rats, Inbred Strains , Silver , Staining and Labeling , Transcription, Genetic
18.
J Virol ; 65(3): 1589-95, 1991 Mar.
Article in English | MEDLINE | ID: mdl-1847476

ABSTRACT

In cells infected with herpes simplex viruses the capsids acquire an envelope at the nuclear membrane and are usually found in the cytoplasm in structures bound by membranes. Infected cells also accumulate unenveloped capsids alone or juxtaposed to cytoplasmic membranes. The juxtaposed capsids have been variously interpreted as either undergoing terminal deenvelopment resulting from fusion of the envelope with the membrane of the cytoplasmic vesicles or undergoing sequential envelopment and deenvelopment as capsids transit the cytoplasm into the extracellular space. Recent reports have shown that (i) wild-type virus attaches to but does not penetrate cells expressing glycoprotein D (G. Campadelli-Fiume, M. Arsenakis, F. Farabegoli, and B. Roizman, J. Virol. 62:159-167, 1988) and that (ii) a mutation in glycoprotein D enables the mutant virus to productively infect cells expressing the wild-type glycoprotein (G. Campadelli-Fiume, S. Qi, E. Avitabile, L. Foa-Tomasi, R. Brandimarti, and B. Roizman, J. Virol. 64:6070-6079, 1990). If the unenveloped capsids in the cytoplasm result from fusion of the cytoplasmic membranes with the envelopes of viruses transiting the cytoplasm, cells infected with virus carrying the mutation in glycoprotein D should contain many more unenveloped capsids in the cytoplasm inasmuch as there would be little or no restriction in the fusion of the envelope with cytoplasmic membranes. Comparison of thin sections of baby hamster kidney cells infected with wild-type and mutant viruses indicated that this was the case. Moreover, in contrast to the wild-type parent, the mutant virus was not released efficiently from infected cells. The conclusion that the unenveloped capsids are arrested forms of deenveloped capsids is supported by the observation that the unenveloped capsids were unstable in that they exhibited partially extruded DNA.


Subject(s)
Capsid/genetics , Cell Transformation, Viral , Simplexvirus/genetics , Viral Envelope Proteins/genetics , Animals , Capsid/ultrastructure , Cell Line , Cytoplasm/ultrastructure , Kinetics , Microscopy, Electron , Mutation , Simplexvirus/ultrastructure , Time Factors
19.
Clin Exp Immunol ; 83(2): 291-7, 1991 Feb.
Article in English | MEDLINE | ID: mdl-1993361

ABSTRACT

The specificities of anti-nuclear antibodies (ANA) reacting with multiple nuclear dots (MND-ANA) present in about 15% primary biliary cirrhosis sera were studied by Western blot analysis with nuclear fractions from a human cell line. Reactivity with two broad bands of 78-92 kD and 96-100 kD of the insoluble fraction was present exclusively in MND-ANA-positive sera. Antibodies eluted from these proteins specifically retained the immunofluorescence reactivity of MND-ANA. Immunomorphological analysis by a pre-embedding technique revealed that the antibody specifically binds to nuclear regions resembling in size and number nuclear bodies. Since these structures are absent in immature rate endometrial cell and can be induced by diethylstilbestrol, we tested MND-ANA by immunofluorescence on cryostat sections of uteri from hormone-treated and untreated immature rats. A strong reaction of nuclear dots was observed predominantly in endometrial cells of hormone treated rats. We thus conclude that MND-ANA present in primary biliary cirrhosis sera are directed against 78-92-kD and 96-100-kD nuclear proteins located in nuclear bodies.


Subject(s)
Antibodies, Antinuclear/immunology , Liver Cirrhosis, Biliary/immunology , Nuclear Proteins/analysis , Animals , Blotting, Western , Female , Fluorescent Antibody Technique , Humans , Microscopy, Immunoelectron , Rats , Rats, Inbred Strains , Uterus/chemistry
20.
J Histochem Cytochem ; 38(10): 1495-501, 1990 Oct.
Article in English | MEDLINE | ID: mdl-2205645

ABSTRACT

Osmium-ammine (OA)/SO2 selectively contrasted RNA- and DNA-containing structures in thin sections from Lowicryl-embedded samples. No cell structures were stained after Epon embedding. RNAse and DNAse digestion experiments demonstrated that only RNA and DNA were stained in Lowicryl thin sections. Protease digestion did not modify the staining reaction. The very fine end-reaction produced a very high resolution of the stained structures. The staining reaction was not due to the presence of SO2 but to the low pH of the solution (ranging from 1.5-2.2). OA in glycine buffer, pH 1.5, selectively contrasted nucleic acids. Electrostatic bonds between nucleic acids and OA complex were probably involved in the staining reaction. Increasing the pH value of the staining medium resulted in loss of OA specificity for nucleic acids. The high electrolyte concentration of the staining medium hindered the staining reaction.


Subject(s)
Histocytochemistry/methods , Microscopy, Electron/methods , Nucleic Acids/metabolism , Osmium Compounds , Acrylic Resins , Animals , Carcinoma, Ehrlich Tumor/metabolism , Carcinoma, Ehrlich Tumor/pathology , Carcinoma, Ehrlich Tumor/ultrastructure , Deoxyribonucleases/pharmacology , Humans , Hydrogen-Ion Concentration , Kidney/metabolism , Kidney/pathology , Kidney/ultrastructure , Lymphocytes/metabolism , Lymphocytes/ultrastructure , Mice , Nucleic Acids/drug effects , Nucleic Acids/ultrastructure , Osmium , Peptide Hydrolases/pharmacology , Quaternary Ammonium Compounds , Rats , Ribonucleases/pharmacology
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