p53 and p21waf-1 expression correlates with apoptosis or cell survival in poorly differentiated, but not well-differentiated, retinoblastomas.

In human retinoblastomas, rare genetic mutations of the retinoblastoma gene cause massive cell proliferation, altered differentiation, and tumor formation; but paradoxically, this is accompanied by extensive apoptotic cell loss. We quantified the immunohistochemical distribution of p53, its downstream effector p21 (WAF-1), and apoptotic cells in 50 human retinoblastomas, within three concentric zones of sleeves of tumor cells surrounding blood vessels. In poorly differentiated retinoblastomas, both p53 expression and apoptosis increase toward the outer zone of tumor sleeves, whereas p21 expression occurs primarily within the inner zone. This staining pattern of p53 expression is reversed in well-differentiated tumors, whereas p21 staining and apoptotic cell distributions are unchanged. We detected no p53 mutations in four retinoblastomas and two retinoblastoma cell lines. We postulate that oxygen and cell "survival/growth factors" delivered via blood vessels protect retinoblastoma cells from apoptosis. In poorly differentiated tumors, apoptosis is spatially associated with increased p53 expression and may be p53 mediated, but in well-differentiated tumors, apoptosis does not colocalize with p53 and may be p53 independent. In retinoblastomas, p21 is involved not in cell death by apoptosis but in cell survival. Thus, p53 varies its expression (and by implication its function) with altered differentiation in retinoblastomas.

Bisphosphonates induce apoptosis in mouse macrophage-like cells in vitro by a nitric oxide-independent mechanism.

Bisphosphonates (BPs) are an important class of antiresorptive drugs used in the treatment of bone diseases, including osteoporosis. Although their mechanism of action has not been identified at the molecular level, there is substantial evidence that BPs can have a direct effect on osteoclasts by mechanisms that may lead to osteoclast cell death by apoptosis. BPs can also inhibit proliferation and cause cell death in macrophages in vitro. We have now shown that the toxic effect of BPs on macrophages is also due to the induction of apoptotic, rather than necrotic, cell death. Morphological and biochemical features that are definitive of apoptosis (chromatin condensation, nuclear fragmentation, and endonuclease-mediated internucleosomal cleavage of DNA) could be identified in mouse macrophage-like J774 and RAW264 cells, following treatment with 100 microM pamidronate, alendronate, and ibandronate for 24 h or more. Clodronate was much less potent, even at 2000 microM, while 2000 microM etidronate did not cause apoptosis. Apoptosis was not due to increased synthesis of nitric oxide and could not be prevented by inhibitors of nitric oxide synthases. Since macrophages, like osteoclasts, are particularly susceptible to BPs, these observations support the recent suggestion that the mechanism by which BPs inhibit bone resorption may involve osteoclast apoptosis. Furthermore, the macrophage-like cell lines used in this study may be a convenient model with which to identify the molecular mechanisms by which BPs promote apoptosis in osteoclasts. Induction of macrophage apoptosis by BPs in vivo may also account, at least in part, for the anti-inflammatory properties of BPs as well as the ability of BPs to cause an acute phase response.

A study of death by anoikis in cultured epithelial cells.

BACKGROUND: Epithelial cells are critically dependent upon cell-matrix and cell-cell adhesion for growth and survival. Anoikis is programmed cell death caused by disruption of cell-substrate adhesion in normal epithelial cells. METHODS: We studied the induction of anoikis in vitro in two cell lines; HaCaT and SW742. PI3K, JAK2 and PKC are key elements in signalling pathways regulating cell survival, and using specific inhibitors we also examined their potential role in the induction of anoikis. RESULTS: When prevented from adhesion by culture on polyHEMA, HaCaT cells underwent apoptosis selectively from the proliferating population; surviving cells underwent cell cycle arrest. In SW742 cells anoikis also occurred, but was balanced by increased cycling. The effects of specific kinase inhibitors indicated that both Janus kinase 2 and protein kinase C partially protect HaCaT cells from anoikis through inducing cell cycle arrest of surviving nonadherent cells; inhibition of Phosphatidylinositol 3-kinase did not induce cycling in HaCaTs prevented from adhesion but did stimulate anoikis. SW742 cells showed markedly different responses: Janus kinase 2 inhibition activated apoptosis directly, Phosphatidylinositol 3-kinase inhibition stimulated both cell cycling and apoptosis, while protein kinase C inhibition stimulated cycling but inhibited apoptosis. CONCLUSIONS: Susceptibility to cell death in adhesion-prevented epithelial cells may thus be regulated by signalling pathways involving Phosphatidylinositol 3-kinase, Janus kinase 2 and protein kinase C. The ability of epithelial tumour cells to invade and metastasize may therefore result from disruption of these pathways.

Cell cycle kinetics, tissue transglutaminase and programmed cell death (apoptosis).

Studies were undertaken on a highly metastatic hamster fibrosarcoma cell line with a view to assessing whether cells entering into apoptosis, measured by counting the number of transglutaminase mediated detergent insoluble envelopes, has any synchrony with a particular phase of the cell cycle. A double exposure of thymidine was used to block cells in early S-phase. Flow cytometry in combination with [3H]thymidine incorporation into DNA was used to assess the degree of synchrony and progression through the different phases of cell cycle. The apoptotic index was found to be at its maximum in mid-S-phase. Measurement of transglutaminase activity in each phase of the cell cycle indicated that the specific activity was also at its greatest during mid S-phase. The level of enzyme was relatively unchanged throughout the cell cycle indicating that the regulation of transglutaminase activity occurs primarily through effects on catalytic activity rather than enzyme synthesis.

The importance of the GTP-binding protein tissue transglutaminase in the regulation of cell cycle progression.

Tissue transglutaminase (tTgase) is a GTP-binding Ca(2+)-dependent enzyme which catalyses the post-translational modification of proteins via epsilon(gamma-glutamyl) lysine bridges. Recent evidence suggests that the GTP-binding activity of tTgase may be important in intracellular signaling thus explaining some of the diverse suggested roles for the enzyme. In the following work a malignant hamster fibrosarcoma (Met B) has been stably transfected with both the full length tTgase cDNA (wild type) and a mutant form of the cDNA whereby the active site cysteine (Cys 277) has been replaced by serine. Expression of this mutant cDNA leads to a protein with GTP binding activity which is deficient of protein crosslinking activity. When synchronised into S-phase and allowed to progress through the cell cycle tTgase transfected clones (both mutant and wild type), when compared to transfected controls, show a delayed progression from S-phase to G2/M when analysed by flow cytometry which appears to be elicited by the G-protein activity of the tTgase.

The identification of informative parameters in the flow cytometric analysis of breast carcinoma.

DNA ploidy and the measurement of proliferation or S-phase fraction are both of prognostic significance in breast cancer, yet clinical use is minimal in the U.K. Immunohistochemistry is, however, used to aid diagnosis, so a panel of antibodies were analysed by flow cytometry to assess their predictive value for prognosis, tumour stage and grade. Of 10 parameters tested on 202 breast tumour samples, tumour cell proliferation and DNA ploidy were the two most informative; cytokeratin staining, natural killer and B-cell infiltration also proved to be of value but there was no prognostic value in measuring tumour infiltrating monocytes, helper/suppressor T-cell ratios, tumour cell reactivity with carcinoembryonic antigen or human milk fat globulin antibodies. For each of the informative parameters, scores numerically weighted towards a poorer prognosis were derived which when combined, correlated with tumour grade, stage and prognosis. Such data interpretation is objective, and can be transposed to other human tumours.

Megakaryocyte and vascular changes in rabbits on a short-term high cholesterol diet.

The effect of a short-term high cholesterol diet on thrombopoiesis and vascular ultrastructure was evaluated in rabbits. Six pairs of male litter-mate rabbits were randomized pairwise to feeding with either 2 g of cholesterol daily in addition to their normal diet or normal diet alone for 7 days. A significant 12-fold increase in median serum cholesterol (P less than 0.035) and an insignificant decrease in platelet count (P = 0.07) were found in the animals fed a high cholesterol diet. In these animals the total and cytoplasmic megakaryocyte size measured as planimetric areas in bone marrow sections were significantly decreased (P less than 0.035). No statistically significant difference in the megakaryocyte DNA content measured by flow cytometry in marrow suspensions enriched for megakaryocytes by density gradient centrifugation and centrifugal elutriation was observed between the cholesterol-fed animals and controls. Light microscopic, transmission and scanning electron microscopic examination of the aorta in both groups of animals showed a morphologically intact endothelium without any adhesion of blood-borne cells to the luminal surface. Transmission electron microscopic studies showed that cells with ultrastructural features resembling smooth muscle cells were present in the intima of the aortas of the animals on the high cholesterol diet, but not in control animals. A decrease in the size of bone marrow megakaryocytes and the occurrence of intimal smooth muscle cells are found in rabbits fed a high cholesterol diet for 7 days. These cellular events may be important features in early atherogenesis.

The detection of intracytoplasmic interleukin-2 in Jurkat E6.1 and human peripheral blood mononuclear cells using direct conjugate, two-colour, immunofluorescent flow cytometry.

The most commonly used approaches for the estimation of cytokine protein production involve the quantification of cytokines produced, and accumulated, in a complex body fluid or supernatant of cultured cells, by means of a bioassay or immunoassay, but these techniques do not permit an estimation of the frequency or phenotype of cytokine-producing cells. Traditional methods use immunohistochemical based techniques which can be difficult to perform and interpret, whereas flow cytometry has the advantage of objective assessment and standardisation and is less labour intensive. In this study we have established a rapid and sensitive technique for the simultaneous detection of intracellular IL-2 in conjunction with CD3. Polyclonal goat anti-IL-2 and control goat IgG were conjugated to FITC and then separated from free fluorochrome using column chromatography. PHA activated PBMC or Jurkat E6.1 cells were fixed in paraformaldehyde and permeabilised with saponin, followed by the addition of directly conjugated antibodies (FITC anti-IL-2 or PE anti-CD3) alone or in combination. Samples were then analysed using a flow cytometer and the percentage of dual labelled cells calculated. Several methods have been previously established for the detection of intracellular cytokines using flow cytometry and employing multiple layers of antibodies in the detection steps. By using direct conjugates the technique is less time consuming, requires fewer controls and can be used to examine cytokine production by identifiable cell phenotypes in a mixed cell population.

DN 9693: a phosphodiesterase inhibitor with a platelet membrane effect.

We have examined the in vitro effects of DN 9693 (piperidinylimidazo-quinazolinone) on various aspects of platelet reactivity. Our results are consistent with its known function as a phosphodiesterase inhibitor in that it increased platelet cyclic AMP, particularly in conjunction with an adenylate cyclase stimulator, and exerted a profound inhibitory effect on platelet aggregation responses to a variety of agonists. DN 9693 also inhibited ristocetin-induced platelet agglutination (RIPA). We therefore examined its effect on ristocetin co-factor assays and on the binding of a monoclonal antibody (McAb) to platelet membrane glycoprotein Ib (GPIb). The drug inhibited the binding of the monoclonal antibody in a dose-dependent manner. This suggests an effect of the drug on the platelet surface membrane with reduced expression of GPIb. Our results indicate that in addition to its anticipated inhibitory effect on platelet aggregation, DN 9693 may also inhibit platelet adhesion.

TNF alpha protects SW742 human colon carcinoma cells against non-MHC-restricted cytolysis.

The ability of interferons (IFN alpha and IFN gamma) to protect human tumour cells from non-MHC-restricted cytotoxicity is well established. We show that in addition to rhIFN gamma, rhTNF alpha is also able to decrease the susceptibility of the human colon carcinoma cell line SW742 to non-MHC-restricted lysis by fresh and IFN alpha activated peripheral blood mononuclear cells. The observed decrease in lysis was not the result of a decrease in the rate of killing. rhTNF alpha and rhIFN gamma were unable to alter MHC class 1 expression, indicating that the protection induced was not the result of increased class 1 antigen expression; however, both cytokines enhanced ICAM-1 expression on the tumour cells. rhTNF alpha and rhIFN gamma did not alter the proliferation or cell-cycle profile of SW742 cells, indicating that the protection was not cell-cycle phase dependent and was not secondary to suppression of cell growth.

Interleukin-6 and tumour necrosis factor alpha synergistically block S-phase cell cycle and upregulate intercellular adhesion molecule-1 expression on MCF7 breast carcinoma cells.

The effect of IL-6 and TNF alpha were studied on human MCF7 breast cancer cells. Synergistic interaction between IL-6 and TNF alpha, on the growth inhibition (50% reduction in the percentage of S-phase cells) and the upregulation of ICAM-1 expression (4 to 11-fold increase) was shown using flow cytometric methods. IL-6 and TNF alpha alone had negligible effect on the cell cycle. The individual effect of IL-6 resulted in down-regulation of ICAM-1 expression (30-35%), while TNF alpha always upregulated ICAM-1 (1.5 to 4-fold increase). The combined effect of IL-6 and TNF alpha consistently caused an increased expression of ICAM-1, which was greater than the sum of each one alone and also sustained for 72 h following cytokine withdrawal.

2D-gel analysis of proteins in chronic lymphocytic leukemia cells and normal B-lymphocytes.

Protein synthesis was analysed in leukaemic cells from 10 chronic lymphocytic leukaemia (CLL) patients by 2D-gel electrophoresis of 14C-labelled proteins. There appeared to be only minor differences between each of the CLL samples, but there was evidence that the level of expression of a few of the proteins might have correlated to the stage of the disease. Comparison of the CLL samples to populations of normal B-lymphocytes demonstrated marked differences in protein synthesis between the leukaemic and non-malignant cells. We subsequently used the fluorescence activated cell sorter (FACs) to separate CD5+ from CD5- B-lymphocytes, but observed that the protein synthesis exhibited by these two populations was essentially the same, and both were very different to that observed in CLL cells. The significance of these observations with respect to the origins of CLL is discussed.

The prediction of recurrence in meningiomas. A flow cytometric study of paraffin-embedded archival material.

Despite the complete macroscopic excision of meningiomas, there is a significant rate of recurrence approaching 20% at 20 years. The prediction of recurrence by clinical and histopathological means is inadequate. Flow cytometric analysis of deoxyribonucleic acid (DNA) in meningiomas has shown a correlation between a high proliferative index based on tumor cell-cycle stage (%S + %G2/M) and clinically aggressive behavior. Accordingly, the DNA analysis of meningioma tissue may be of value in predicting recurrence of these tumors. To test this hypothesis, the DNA of paraffin-embedded archival tissue from known recurrent meningiomas was compared with an age- and sex-matched nonrecurrent group. Both groups had comparable follow-up periods. Forty patients with total macroscopic removal at the time of surgery were analyzed. The paraffin blocks of these tumors were retrieved and reclassified histologically according to the World Health Organization system. Sections were then taken for flow cytometric study. The DNA analysis showed that the proliferative index of the recurrent group was significantly higher than that of the nonrecurrent group (p less than 0.002), although the histological subtyping of the two groups was similar. These results support the suggestion that flow cytometry may be of value in the prediction of recurrence of histologically benign, macroscopically removed meningiomas.

DNA in meningioma tissues and explant cell cultures. A flow cytometric study with clinicopathological correlates.

Flow cytometry was performed on stored frozen tissues and explant cell cultures from 39 meningiomas using ethidium bromide and mithramycin in a selective staining technique for deoxyribonucleic acid (DNA). The ploidy index and percentage of cells in the G0/G1, S, and G2/M phases were calculated for each specimen. The results were compared with the age and sex of the patients; the site, the histological subtype, and mitotic rate of the neoplasms; and the estrogen- and progesterone-receptor levels assayed in cytosol-enriched supernatants from cryostat-cut sections. Sixteen neoplasms (41%) were aneuploid. These included two recurrent neoplasms, seven of the eight neoplasms from patients with multiple meningiomas, and three clinically aggressive neoplasms (one hemangiopericytic and two anaplastic meningiomas). Significant correlations were found between values for the ploidy index (r = 0.75, p less than 0.01), the percentage of S-phase cells (r = 0.82, p less than 0.01), and the percentage of G2/M-phase cells (r = 0.69, p less than 0.05) in vivo and in vitro. The results support the suggestion that flow cytometry for DNA in meningiomas may be of value in predicting the behavior of these neoplasms, and indicate that under controlled conditions explant cell cultures may provide a useful model for the proliferative characteristics of meningiomas in vivo.

Cytokine modulation of antigen expression in human melanoma cell lines derived from primary and metastatic tumour tissues.

The constitutive and cytokine-mediated expression of MHC class I and II antigens and intercellular adhesion molecule-1 (ICAM-1) was evaluated on eight human melanoma cell lines derived from primary and metastatic malignancies from patients (WM human melanoma series) including three pairs of related cell lines derived from the same individual. The cytokines IL-1 beta, IL-4, IL-6, TNF alpha, TGF beta 2, IFN gamma and IFN alpha were assessed for their ability to modulate the expression of cell surface antigens. MHC class I and class II antigen expression was unregulated by IFN gamma, IFN alpha and/or TNF alpha in cell lines established from primary melanoma. In contrast the cell lines derived from metastatic deposits did not show an increase in expression of MHC antigens in response to these cytokines. Both primary and metastatic WM cell lines were shown to be resistant to spontaneous natural killer cell (NK) activity, but susceptible to effector lymphocytes mediating lymphotine activated killer (LAK) cytotoxicity as a result of activation by IL-2. Although the constitutive and cytokine-induced level of expression of ICAM-1 and MHC antigens varied between paired primary and metastatic cell lines, this did not correlate with susceptibility of the cell line target to NK or LAK cytotoxicity. Whereas the IFNs, TNF alpha, TGF beta 2 and IL-1 beta differentially modulated the expression of ICAM-1 and MHC class I, treatment with IFNs (but not IL-1 beta, TNF alpha or TGF beta 2) resulted in a significant reduction in the sensitivity of the melanoma cells to NK and LAK cytotoxicity. Constitutive ICAM-1 expression was positively correlated with the ability of WM cell lines to colonise the lungs of SCID mice upon i.v. injection. The acquisition of cytokine resistance and inability to demonstrate enhanced cell surface expression may represent an important feature associated with the development of the metastatic phenotype.

On the mechanism of the post-asymptotic CR decrement phenomenon.

Three experiments with 49 dogs explored the decrease in CR magnitude that sometimes occurs when CS-US pairings are continued beyond those needed to reach maximum CR magnitude. The first experiment confirmed the existence of the phenomenon, obtaining less conditioned excitation after 300 CS-US trials than after 18 CS-US trials. The second experiment demonstrated that the phenomenon is not dependent upon paired CS-US presentations because 18 CS-US pairings yielded little excitation if preceded by, followed by, or intermixed with 282 US presentations. The third experiment indicated that in contrast to the decremental effects on excitatory conditioning, inhibitory conditioning was faciliated by large numbers of prior US exposures, suggesting explanation of the post-asymptotic decrement phenomenon by opponent-process theory.

Extracting textural features from tactile sensors.

This paper describes an experiment to quantify texture using an artificial finger equipped with a microphone to detect frictional sound. Using a microphone to record tribological data is a biologically inspired approach that emulates the Pacinian corpuscle. Artificial surfaces were created to constrain the subsequent analysis to specific textures. Recordings of the artificial surfaces were made to create a library of frictional sounds for data analysis. These recordings were mapped to the frequency domain using fast Fourier transforms for direct comparison, manipulation and quantifiable analysis. Numerical features such as modal frequency and average value were calculated to analyze the data and compared with attributes generated from principal component analysis (PCA). It was found that numerical features work well for highly constrained data but cannot classify multiple textural elements. PCA groups textures according to a natural similarity. Classification of the recordings using k nearest neighbors shows a high accuracy for PCA data. Clustering of the PCA data shows that similar discs are grouped together with few classification errors. In contrast, clustering of numerical features produces erroneous classification by splitting discs between clusters. The temperature of the finger is shown to have a direct relation to some of the features and subsequent data in PCA.

A hydrostatic Michell framework supports frog lungs.

A braced framework of tubular struts, in the walls and air spaces of frog lungs, suspends the respiratory surface and holds the lung open at zero transmural pressure withstanding imploding forces created by abdominal viscera, much as would the supports of a bell tent. The struts are tubes, having a larger second moment of area than do solid struts of the same cross-sectional area, and so are stronger, and contain pulmonary vessels within a flexible wall. The orthogonal arrangement of the struts in the framework, explained in part by Maxwell's Lemma and Michell's Theorem, strengthens the framework and minimizes its weight; orthogonality is maintained as the lungs change size. A model is presented, in which a frog might control pre- and post-pulmonary vascular resistances and, hence, blood volume in the struts, without compromising pulmonary perfusion. Such adjustments could vary the area of lung and the extent of perfused capillaries exposed to pulmonary gas, helping match the lung's surface area, weight and metabolic load to activity.

Evidence that interleukin-4 suppression of lymphokine-activated killer cell induction is mediated through monocytes.

Recombinant human interleukin-4 (IL-4) and transforming growth factor-beta (TGF-beta) reduce recombinant interleukin-2 (IL-2) induction of lymphokine-activated killer (LAK) cell activity from human peripheral blood mononuclear cells (PBMC). Monocytes can be removed from PBMC by adherence, leaving a peripheral blood lymphocyte population (PBL) which also responds to IL-2 to generate LAK activity. PBL generation of LAK cytotoxicity is susceptible to inhibition by TGF-beta, but not by IL-4. Readdition of purified monocytes to PBL is accompanied by return of the suppressive action of IL-4 on the generation of LAK activity. Induction of LAK cytolysis from Percoll-isolated T cells (greater than 90% CD3+) is also refractory to the inhibitory effect of IL-4. When PBMC were cultured in IL-2, with and without IL-4, subsequent sorting of CD3+ and CD3- lymphocytes by flow cytometry demonstrated that IL-4 had suppressed LAK induction in both effector populations. This suggests that, although isolated CD3+ cells are not susceptible to IL-4 suppression of IL-2 activation, they are sensitive to inhibition when part of a mixed PBMC population. Evidence is presented for the first time that this suppression is mediated via the action of IL-4 on monocytes.