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W Gorczyca

Publications and source records attributed to W Gorczyca.

At least 37 records · Page 2Linked to original sources
PubMed1997

Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis).

The term cell necrobiology is introduced to comprise the life processes associated with morphological, biochemical, and molecular changes which predispose, precede, and accompany cell death, as well as the consequences and tissue response to cell death. Two alternative modes of cell death can be distinguished, apoptosis and accidental cell death, generally defined as necrosis. The wide interest in necrobiology in many disciplines stems from the realization that apoptosis, whether it occurs physiologically or as a manifestation of a pathological state, is an active mode of cell death and a subject of complex regulatory processes. A possibility exists, therefore, to interact with the regulatory machinery and thereby modulate the cell's propensity to die in response to intrinsic or exogenous signals. Flow cytometry appears to be the methodology of choice to study various aspects of necrobiology. It offers all the advantages of rapid, multiparameter analysis of large populations of individual cells to investigate the biological processes associated with cell death. Numerous methods have been developed to identify apoptotic and necrotic cells and are widely used in various disciplines, in particular in oncology and immunology. The methods based on changes in cell morphology, plasma membrane structure and transport function, function of cell organelles, DNA stability to denaturation, and endonucleolytic DNA degradation are reviewed and their applicability in the research laboratory and in the clinical setting is discussed. Improper use of flow cytometry in analysis of cell death and in data interpretation also is discussed. The most severe errors are due to i) misclassification of nuclear fragments and individual apoptotic bodies as single apoptotic cells, ii) assumption that the apoptotic index represents the rate of cell death, and iii) failure to confirm by microscopy that the cells classified by flow cytometry as apoptotic or necrotic do indeed show morphology consistent with this classification. It is expected that flow cytometry will be the dominant methodology for necrobiology.

Apoptosis↗
PubMed1997

Laser scanning cytometric analysis of cyclin B1 in primary human malignancies.

Cyclins are key components of the cell cycle progression machinery. They activate their partner-dependent kinases (CDKs) and target them to respective substrate proteins within the cell. CDK-mediated phosphorylation of specific sets of proteins drives the cell through particular phases or checkpoints of the cell cycle. During unperturbed growth of normal cells, the timing of expression of several cyclins is discontinuous, occurring at discrete and well-defined periods of the cell cycle. Immunocytochemical detection of cyclins in relation to cell cycle position (DNA content) by multiparameter flow cytometric techniques has provided a new approach to cell cycle studies. This approach, like no other method, can be used to detect the "unscheduled" expression of cyclins, namely, the presentation of G1 cyclins by cells in G2/M and of G2/M cyclins by G1 cells, without the need for cell synchronization. By use of multiparameter flow cytometric and laser scanning cytometric analysis, we correlated the expression of cyclin B1 with cell cycle position in normal lymphocytes stimulated to proliferate by the mitogen phytohemagglutinin and in 28 primary human tumors of different organ and type. Eighteen of the 28 tumors expressed the cyclin B1 in more than 5% of cells (B1 positive), and the rest showed cyclin expression from 2.1 to 5% (B1 negative). In normal lymphocytes, the expression of cyclin B1 was restricted to very late S and G2 + M phases of the cell cycle. In 15 of 18 primary tumors studied, the expression of cyclin B1 was "unscheduled" (unrestricted to particular phases of the cycle). The data suggest that the "unscheduled" expression of cyclin B1 might be a common defect in neoplasia.

Adrenal Gland Neoplasms↗
PubMed1997

[Retinal calcium-binding proteins--their function and pathology].

Calcium is involved in most cellular processes through calcium-binding proteins, belonging to the EF-hand superfamily. Recently new members of this family were found in the retina of vertebrates. Functions of these proteins and their relations to some neurological diseases are reviewed. Other EF-hand proteins found in the retina are also described.

Animals↗
PubMed1996

Mapping of the basal forebrain cholinergic system of the dog: a choline acetyltransferase immunohistochemical study.

In an effort to produce a canine model of basal forebrain ischemia with memory deficits, we have shown that dogs possess a medial striate artery that perfuses basal forebrain territory, homologous to the human recurrent artery of Heubner. In the present study, we set out to delineate the precise topography of the cholinergic neurons in the canine forebrain, a neuronal system implicated in cognitive and memory functions. Floating coronal sections, derived from the head of the caudate nucleus to the rostral border of the hippocampus, were stained for choline acetyltransferase using a monoclonal antibody. Representative sections from one dog brain were drawn. These outlines were used for measurement of cell density, cell size, number of processes, and cell roundness. Choline acetyltransferase-positive neurons constituted four major subdivisions within the basal forebrain. A relatively dense population of cholinergic neurons was present in the medial septal nucleus (Ch1). A continuum of densely packed cells was also delineated within the vertical (Ch2) and horizontal (Ch3) nuclei of the diagonal band of Broca. A fourth group of heterogeneously packed cholinergic neurons represented the nucleus basalis magnocellularis (Ch4). Except for the caudal component of the Ch4 population, the forebrain cholinergic corticopetal system was located within the perfusion territory of the medial striate arteries. The Ch4 cell group in dogs is better defined than that of rodents but is not as sharply demarcated as in human and nonhuman primates. Our findings indicate that the dog may serve as an excellent model for assessing neurological and memory deficits, which, in humans, results from hypoperfusion of the recurrent artery of Heubner.

Analysis of Variance↗
PubMed1996

Microvascular anatomy of striate vessels in dogs: contribution to an experimental model of forebrain ischemia.

Basal perforating vessels such as Heubner's artery are involved in various arteriopathic conditions resulting in cognitive and memory deficits. In order to evaluate if a model of focal forebrain ischemia can be obtained in dogs, a systematic study of the microvascular anatomy of the striate vessels was performed using intravascular injections of acrylic paint and/or India ink. Medial and lateral striate arteries were identified and found distinct either by their superficial anatomy as well as their deep perfusion territories. One medial striate artery emerging consistently from the junction of the A1 segment of the anterior cerebral artery and the ethmoidal artery resembles the human recurrent artery of Heubner. Therefore, the dog may provide a suitable model of forebrain ischemia involving solely the perfusion territory of the medial striate arteries.

Anatomy, Artistic↗
PubMed1996

Laser scanning cytometer (LSC) analysis of fraction of labelled mitoses (FLM).

In this report we describe the successful application of a novel microscope-based multiparameter laser scanning cytometer (LSC) to measure duration of different phases of cell cycle in HL-60 human leukaemic cell lines by the fraction of labelled mitoses (FLM) method. Exponentially growing cells were harvested after various time intervals following pulse-labelling with 5'-bromo-2'-deoxyuridine (BrdUrd), cytocentrifuged, fixed in ethanol, and then exposed to UV light to induce DNA strand breaks at the sites of incorporated BrdUrd. The 3' OH termini of the photolytically generated DNA strand breaks were labelled with BrdUTP in the reaction catalysed by exogenous terminal deoxynucleotidyl transferase (TdT), followed by FITC-labelled BrdUrd antibodies. DNA was counterstained with propidium iodide (PI). Due to differences in chromatin structure between the interphase and mitotic cells, the LSC identified the latter by virtue of their higher red (PI) fluorescence intensity values among all pixels over the measured cell. To confirm that the cells selected were indeed cells in mitosis, predominantly in metaphase, the recorded X-Y coordinates of selected cells were used to re-position the cell for their visual examination. From the time lapse analysis of percentage BrdUrd-labelled cells progressing through mitosis it was possible to calculate the duration of individual phases of the cell cycle. The duration of S (Ts) and G2 + M (TG2 + M) was 8 and 3 h, respectively, and the minimal duration of G2 (TG2) was 2 h. The cell cycle time (Tc) estimated for the cohort of the most rapidly progressing cells was 13 h. The ability to automatically and rapidly discriminate mitotic cells combined with the possibility of their subsequent identification by image analysis makes LSC the instrument of choice for the FLM analysis.

Bromodeoxyuridine↗
PubMed1996

Interaction of human erythrocyte glycophorin with immunoglobulins G.

We have found that human glycophorin and asialoglycophorin interacted with human non-immune IgG. To characterize quantitatively the interaction between glycophorin and IgG we elaborated a direct solid-phase radioimmunoassay. We showed that the binding of IgG was reversible and saturable within the range of IgG concentrations used. Glycophorin bound higher amounts of aggregated than non-aggregated immunoglobulins. The apparent association constant for non-aggregated human IgG was 5.45 +/- 0.93 x 10(5) M-1 and 1.13 +/- 0.78 x 10(6) IgG molecules were bound per 1 picogram of the glycophorin. The binding of glycophorin occurs within the F(ab)2 fragment of IgG, mainly.

Asialoglycoproteins↗
PubMed1995

Guinea pig peritoneal macrophages. Differential effects of lectins on interaction with IgG immunoglobulins.

Guinea pig peritoneal macrophages have on their surface two receptors, one (Fc gamma 1/gamma 2 R) binding both guinea pig IgG1 and IgG2 and the second (Fc gamma 2R) binding only IgG2 immunoglobulins. We have previously shown that treatment of macrophages with neuraminidase or glycosylation inhibitors affects, in a different way, the binding of guinea pig IgG1, IgG2, and rabbit IgG. In the present study we have shown that pretreatment of guinea pig macrophages with lectins (Con A, WGA, and PNA) also has a different effect on the interaction of the cells with IgG. The lectins increased the binding of guinea pig IgG1, whereas rabbit IgG and guinea pig IgG2 were bound with a lower efficiency than in the case of control cells. Since sialic acid residues seem to modulate the activity of receptors and WGA interacts with sialylated oligosaccharides, we determined the IgG-binding characteristics for WGA-pretreated macrophages. We found that the increase in IgG1-binding ability was caused by an increase in the value of Kapp, but the number of IgG-binding sites was lower than in the control cells. In the case of rabbit IgG and guinea pig IgG2 we observed a decrease of both the value of Kapp and the number of IgG-binding sites. WGA did not interact directly with the Fc gamma receptor. The results of our former papers and the different effects of lectins of various specificities described in this paper suggest different positions of Fc gamma 1/gamma 2 and Fc gamma 2R in the plane of the macrophage membrane in respect to various membrane glycoconjugates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Immunohistochemical analysis of bcl-2 and p53 expression in breast carcinomas: their correlation with Ki-67 growth fraction.

We examined 59 breast cancers for p53 and bcl-2 protein expression by immunohistochemistry. The results were correlated with Ki-67 immunostaining. p53-negativity was noted in 40 cases and the remaining 19 tumours were p53-positive. Thirty-six tumours showed strong expression of bcl-2 and in 23 no staining for this protein was observed. We found statistically significant reverse correlation between expression of p53 and bcl-2 in majority of carcinomas: 31 cases were bcl-2 positive and p53-negative, and 14 tumours were bcl-2-negative and p53-positive. Six carcinomas showed no nuclear staining for Ki-67 and in the remaining 53 the percent of cancer cells positive for Ki-67 ranged from 1 to 60 (mean: 14.6). In these 53 cases we found that bcl-2-positive tumours were characterized by lower proliferation than bcl-2-negative tumours, the mean value of Ki-67 immunostaining being 10.7% and 23.0%, respectively. p53-negative tumours showed lower proliferation than p53-positive tumours: mean Ki-67 index was 10.2% and 23.9%, respectively. We conclude that immunohistochemically detected p53 and bcl-2 proteins show a significant inverse relationship in majority of breast carcinomas and their expression correlates with tumour proliferation (Ki-67 immunostaining).

Adult↗
PubMed1995

Chemical synthesis and characterization of ShK toxin: a potent potassium channel inhibitor from a sea anemone.

ShK-toxin, a 35 residue peptide isolated from the sea anemone Stichodactyla helianthus, was synthesized using an Fmoc strategy and successfully folded to the biologically active form containing three intramolecular disulfide bonds. The ability of synthetic ShK toxin to inhibit specific [125I]-dendrotoxin I binding to rat brain membranes slightly exceeded (was more potent than) that of the natural ShK toxin sample, but was comparable with previously reported data for ShK toxin. The peptide toxin inhibited [125I]-charybdotoxin binding to Jurkat T lymphocytes with an IC50 value of 32 pM. In addition, Jurkat T lymphocytes Kv1.3 potassium channels were inhibited with an IC50 value of 133 pM. Owing to their unique structure and high affinity for at least some potassium channels, ShK toxin and related sea anemone potassium channel toxins may become useful molecular probes for investigating potassium channels.

Amino Acid Sequence↗
PubMed1995

Study of microencapsulation for pituitary transplantation: capsule preparation and in vitro study.

We have developed microcapsules using sodium alginate (SA) and Poly-L-Lysine (PLL). A factorial design method of screening was chosen to study influences of different experimental parameters on size and stability of the capsules. We found that air flow affects initial size of the capsules significantly, while the molecular weight (MW) of PLL and incubation time have a positive impact on the expansion when capsules are incubated in sodium citrate (SC). When the capsules were continuously shaken in an attempt to mimic in vivo environmental conditions, those capsules made with optimal parameters (0.1% PLL, 42,000 MW, incubated for 6 minutes; 1.5% SA, incubated for 4 minutes; SC bath 4 minutes; 25# needle; air flow 14L/min) were still intact after 30 days and not totally ruptured until 90 days, while those developed with less strict parameters were ruptured within 2 hours in 50%. We also encapsulated human pituitary adenoma cells using PLL of 80,000 MW and cultured them for 9 days. Adenoma cells, both encapsulated or non-encapsulated, secreted the same amount of hormones. Our preliminary study suggests that selecting optimal combinations of experimental parameters is essential in developing durable microcapsules, which may be potentially used for pituitary transplantation in vivo.

Adenoma↗
PubMed1995

Flow cytometric characterization of proliferation-associated nuclear antigen (p105) during the cell cycle in normal lymphocytes and promyelocytic leukemia cells (HL-60).

Bivariate flow cytometric analysis of p105 expression and DNA content was performed in human lymphocytes and promyelocytic leukemia cells (HL-60). We also employed a new method of detecting DNA strand breaks associated with apoptosis by labeling the 3'-OH termini in the breaks with biotinylated dUTP in a reaction employing exogenous terminal deoxynucleotidyl transferase (TdT). Phytohemagglutin-stimulated proliferating lymphocytes express p105 maximally after 48 hours, similar to HL-60 cells in exponential growth phase. Antigen expression in G1 was notably heterogeneous in G1 phase of both cell types and highest in M-phase cells treated for 6 hours with vinblastine. However, the p105-DNA ratio changed very little. Cycloheximide did not affect P105 expression. Methotrexate decreased p105 expression. Camptothecin and teniposide induced apoptosis, but apoptotic cells still expressed p105. Dual-parameter measurement also demonstrated that TdT-positive apoptotic cells expressed p105 at a higher level than apoptotic TdT negative cells. The data on drug treatment suggest that expression of p105 may be useful in monitoring chemotherapeutic effects but not as a marker of cell death from apoptosis.

Camptothecin↗
PubMed1995

Peanut agglutinin (PNA) binding glycoproteins on human urothelial cell lines of different grades of transformation.

PNA-reactive sites, representing mainly unmasked Thomsen-Friedenreich (TF) antigen, are predictors of invasive transitional cell carcinoma. The present studies were undertaken in order: 1) to quantify the expression of PNA-reactive sites on well-characterized human urinary bladder cell lines belonging to two different transformation grades (TGr II and TGr III); 2) to identify PNA-binding glycoproteins that are restricted in their expression to tumorigenic and invasive urothelial cell lines. Flow cytometry studies revealed significant differences between TGr II and TGr III cell lines. The mean fluorescence intensities of TGr III, tumorigenic and invasive cells, were in the range of 28.4 to 57.3 arbitrary units. The TGr II cells exhibited several fold lower fluorescence intensity: 9.8 arbitrary units for HCV 29 cell line and 13.1 arbitrary units for Hu 609 cells. Neuraminidase treatment, increasing PNA-binding to TGr II as well as in TGr III cell lines, revealed the presence of cryptic PNA-binding sites. The number of cryptic PNA-binding sites seemed to be similar in TGr II and in TGr III cells and, therefore, only add to the total number of PNA-binding sites on native, untreated cells. Binding of 125I-PNA to all cellular proteins resolved by SDS-PAGE and transferred to nitrocellulose showed multiple bands. The TGr III cells lines, after desialylation, were characterized by the presence of two major PNA-binding components represented by diffuse bands with apparent molecular mass about 68-79 kDa and 116-156 kDa, respectively, and two weakly stained bands with apparent molecular mass of 51 kDa and 60 kDa. Both cell lines representing TGr II expressed high molecular mass PNA-binding component of 207 kDa. They were further characterized by the weaker staining intensity of 116-156 kDa glycoproteins as compared to TGr III cell lines.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1994

Detection of apoptosis-associated DNA strand breaks in fine-needle aspiration biopsies by in situ end labeling of fragmented DNA.

The predominant mode of either spontaneous or drug-induced death of cells in tumors is apoptosis. A flow cytometric method was developed in our laboratory to identify apoptotic cells, based on labeling DNA strand breaks, which appear as a result of extensive DNA cleavage by the apoptosis-associated endonuclease, with biotinylated dUTP in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The aim of this study was to reveal whether this methodology can be applied to human solid tumors sampled by fine-needle biopsy. Twenty-two tumors, consisting of 11 breast carcinomas; three metastatic anaplastic carcinomas; three adenocarcinomas of colon, endometrium, and lung; two metastatic lymph node squamous cell carcinomas of the larynx; and three malignant lymphomas were examined. It was possible to identify cells with DNA strand breaks in all these tumors. Extremely high variability in the proportion of cells with DNA strand breaks was observed between the individual tumors. In diploid tumors (n = 12) the percentage of cells with DNA strand breaks varied from 1% to 43%, and the mean value was 19%. In aneuploid tumors this percentage varied from 15% to 51% and the mean value was 37%. In the latter tumors the presence of cells with DNA strand breaks was limited to the DNA aneuploid cell population; very few diploid, presumably tumor infiltrating or stromal cells, showed the presence of DNA strand breaks. No correlation was observed between the percent of cells in S phase and those with DNA strand breaks. The data indicate that apoptosis is more frequent in populations of tumor cells than among normal cells of the same organs.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗
PubMed1994

Apoptosis in adult retinal ganglion cells after axotomy.

Lesions to the mature mammalian central nervous system cause irreversible degeneration, in which neurons have been previously thought to be passive victims. In this study, axon-lesioned adult rat neurons are shown instead to actively degrade themselves through the process of apoptosis: a programmed type of cell death in which the cellular apparatus is actively involved in the degradation process. To investigate whether retinal ganglion cells of an adult mammal follow an apoptotic type of death when their axons are severed, DNA breaks in nuclei were labeled in situ, using a method that specifically incorporates biotinylated deoxynucleotides by exogenous terminal deoxynucleotidyl transferase on the 3'-OH ends of DNA. The active nature of the death mechanism was demonstrated by the reduction in biotin-labeled nuclei after administering the protein synthesis inhibitor cycloheximide. Our results suggest that retinal ganglion cells of the adult rat die through apoptosis when axotomized. This raises new possibilities in the treatment of CNS injuries, by the potential interruptibility of a program for neuronal death.

Animals↗
PubMed1993

The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents.

The studies were aimed to detect the cell cycle-associated differences in the susceptibility of HL-60 cells to apoptosis induced by diverse agents. Exponentially growing HL-60 cells were treated with the DNA topoisomerase I inhibitor camptothecin; the DNA topoisomerase II inhibitors teniposide, m-AMSA, Mitoxantrone, or Fostriecin; the presumed tyrosine kinase inhibitor genistein; a serine/threonine kinase inhibitor H7; the protein synthesis inhibitor cycloheximide; the DNA replication inhibitor hydroxyurea; the nucleoside antimetabolites 1-beta-D-arabinofuranosylcytosine and 5-azacytidine; and the alkylating agent nitrogen mustard, cisplatin, hyperthermia, and gamma irradiation. Endonucleolysis, which accompanied apoptosis induced by these agents, was assessed by two different flow cytometric methods, one based on DNA content measurements following extraction of low molecular weight DNA, and another using exogenous terminal deoxynucleotidyl transferase to label in situ DNA strand breaks. Each method allowed for both identification of apoptotic cells and analysis of the cell cycle distribution of the unaffected cell population; the method using terminal transferase also allowed for identification of the cell cycle position of apoptotic cells. Confirmed by analysis of DNA degradation by gel electrophoresis and changes in cell morphology, apoptosis was observed as early as 3 h after administration of most drugs and for some drugs was cell cycle phase specific. Cells progressing through S phase were selectively susceptible when treated with camptothecin, teniposide, m-AMSA, Mitoxantrone, H7, hydroxyurea, and 1-beta-D-arabinofuranosylcytosine. Cells in G2-M preferentially underwent apoptosis in cultures treated with H7 or with gamma-irradiation. Cells in G1 phase were preferentially affected by 5-azacytidine, nitrogen mustard, and hyperthermia. No significant cell cycle specificity was observed in the case of Fostriecin, genistein, cycloheximide, or cisplatin. The cell cycle related difference in susceptibility to apoptosis may be a reflection of both the severity of the lesion induced by a given drug and the ability of the cells to repair that lesion; both can vary depending on the cell cycle phase.

Antineoplastic Agents↗
PubMed1993

Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays.

DNA strand breaks which occur in HL-60 cells as a result of activation of endonuclease during apoptosis induced by cell treatment with the DNA topoisomerase I inhibitor camptothecin and topoisomerase II inhibitors teniposide, 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide, and fostriecin were labeled in situ, in individual fixed and permeabilized cells, with biotinylated dUTP (detected by fluoresceinated avidin), using the terminal deoxynucleotidyl transferase or nick translation assays. During the early stage of apoptosis, prior to nuclear fragmentation, the breaks were predominantly localized at the nuclear periphery, close to the nuclear envelope. In more advanced stages, all cellular DNA, then localized within the cell as dense, homogeneous granules of a variety of sizes, was strongly labeled, indicating extensive and more uniform distribution of breaks throughout genomic DNA. Bivariate analysis of the incorporated biotinylated dUTP and cellular DNA content by flow cytometry made it possible to estimate the kinetics of the labeling reaction and relate DNA breaks to cell position in the cycle. The kinetics of biotinylated dUTP incorporation was faster, and the distinction of cells with DNA breaks was more pronounced, using the terminal transferase rather than the nick translation assay. Camptothecin, teniposide, and 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide induced DNA breaks preferentially in S-phase cells, having little effect on cells in the G1 phase of the cycle. In contrast, fostriecin affected cells indiscriminately, in all phases of the cell cycle. The method of detection of DNA strand breaks (3'-hydroxyl termini) in individual cells offers several advantages and can be applied to clinical material (tumor biopsies) to study the induction of apoptosis in tumors during treatment, as a possible prognostic marker. The protein-associated DNA breaks in the "cleavable" DNA-topoisomerase complexes, which are the primary lesions induced by the inhibitors and precede apoptosis, were not detectable by the present methods.

Apoptosis↗