A Drosophila PIAS homologue negatively regulates stat92E.

Transcriptional activation by, and therefore the physiologic impact of, activated tyrosine-phosphorylated STATs (signal transducers and activators of transcription) may be negatively regulated by proteins termed PIAS (protein inhibitors of activated stats), as shown by previous experiments with mammalian cells in culture. Here, by using the genetic modifications in Drosophila, we demonstrate the in vivo functional interaction of the Drosophila homologues stat92E and a Drosophila PIAS gene (dpias). To this end we use a LOF allele and conditionally overexpressed dpias in JAK-STAT pathway mutant backgrounds. We conclude that the correct dpias/stat92E ratio is crucial for blood cell and eye development.

Growth and characterization of LNCaP prostate cancer cell spheroids.

Cells from the prostate tumor cell line LNCaP have been grown as spheroids. The growth kinetics of the spheroids have been characterized by fitting a Gompertz equation to spheroid growth curves. The proliferation state of cells within spheroids of different diameters was assessed by bromodeoxyuridine staining. Scanning and electron transmission microscopy were performed to determine the ultrastructure of the spheroids. Prostate-specific antigen (PSA) secretion was monitored throughout spheroid growth. Consistent with Gompertzian kinetics, the volume of LNCaP spheroids initially increased exponentially and then reached a plateau. The doubling time during the exponential phase was 29 +/- 4 h. A core of nonproliferating cells was seen in spheroids with a diameter of 400 microm; at a diameter of 600 microm, a necrotic core had formed. In smaller, 200-microm diameter spheroids, a core of nonproliferating cells was not seen, but proliferating cells were concentrated at the spheroid periphery. Electron microscopy showed that the spheroids were enveloped by an extracellular matrix and that cell adhesion within the spheroids was due in part to desmosomes. PSA secretion by the spheroids could be modeled as originating from a spherical shell whose thickness was independent of overall spheroid diameter. The shell thickness obtained by fitting an appropriate equation to the data was consistent with that determined from the bromodeoxyuridine studies. LNCaP cells exhibit several important features of prostate cancer cells; in vivo, they are androgen responsive, and they express prostatic acid phosphatase, PSA, and prostate-specific membrane antigen. LNCaP spheroids provide a simple but relevant model for the study of drug delivery and response in prostate cancer.

Ykt6p, a prenylated SNARE essential for endoplasmic reticulum-Golgi transport.

Vesicular transport between secretory compartments requires specific recognition molecules called SNAREs. Here we report the identification of three putative SNAREs, p14 (Sft1p), p28 (Gos1p), and a detailed characterization of p26 (Ykt6p). All three were originally isolated as interacting partners of the cis Golgi target membrane-associated SNARE Sed5p, when Sec18p (yeast NSF) was inactivated. YKT6 is an essential gene that codes for a novel vesicle-associated SNARE functioning at the endoplasmic reticulum-Golgi transport step in the yeast secretory pathway. Depletion of Ykt6p results in the accumulation of the p1 precursor (endoplasmic reticulum form) of the vacuolar enzyme carboxypeptidase Y and morphological abnormalities consistent with a defect in secretion. Membrane localization of Ykt6p is essential for protein function and is normally mediated by isoprenylation. However, replacement of the isoprenylation motif with a bona fide transmembrane anchor results in a functional protein confirming that membrane localization, but not isoprenylation per se, is required for function. Ykt6p and its homologues are highly conserved from yeast to human as demonstrated by the functional complementation of the loss of Ykt6p by its human counterpart. This is the first example of a human SNARE protein functionally replacing a yeast SNARE. This observation implies that the specific details of the vesicle targeting code, like the genetic code, are conserved in evolution.

Inhibition of the growth of glioblastomas by CGP 41251, an inhibitor of protein kinase C, and by a phorbol ester tumor promoter.

Protein kinase C (PKC) plays a central role in signal transduction pathways that mediate the action of certain growth factors, tumor promoters, and cellular oncogenes. To explore whether PKC might be an appropriate target for the chemotherapy of human brain tumors, cell lines were established from five glioblastomas, one mixed gliosarcoma and glioblastoma, two astrocytomas, and one choroid plexus carcinoma. The staurosporine derivative CGP 41251, an inhibitor of PKC, inhibited cell proliferation in all nine cell lines with an IC50 in the range of 0.4 micrometer. Drug withdrawal and clonogenicity assays showed that CGP 41251 induced an irreversible growth arrest. Three cell lines were examined in detail: two human glioblastoma cell lines, GB-1 and GB-2, and one gliosarcoma cell line, GS-1. All of these three cell lines were highly aneuploid and displayed morphologies and immunohistochemical markers characteristic of the glial lineage. The compound 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter and activator of PKC, also inhibited the growth of these cell lines. CGP 41251 in combination with TPA caused further growth inhibition. Cultures treated with CGP 41251 displayed an increase in the fraction of cells in G2-M, a decrease of cells in S phase, and no consistent effect on G0-G1. Immunohistochemical analyses demonstrated that growth inhibition by CGP 41251 was associated with the formation of giant nuclei with extensive fragmentation and apoptotic bodies. These effects of CGP 41251 were abrogated by withdrawal of serum from the medium or by exposure of these cells to aphidicolin, actinomycin D, cycloheximide, or TPA. In contrast to the effects seen with the glioblastoma cell lines, nontransformed astrocyte lines remained viable in the presence of 0.4 and 0.8 micrometer CGP 41251 and displayed only a slight increase in the fraction of giant nuclei with fragmentation. The antitumor activity of CGP 41251 was demonstrated in vivo against xenografts of the glioblastoma cell lines U87 MG and U373 MG. These findings suggest that CGP 41251 might be a useful agent for the treatment of glioblastomas.

An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding.

We have isolated a major integral membrane protein from Golgi-derived coatomer-coated vesicles. This 24-kDa protein, p24, defines a family of integral membrane proteins with homologs present in yeast and humans. In addition to sequence similarity, all p24 family members contain a motif with the characteristic heptad repeats found in coiled coils. When the yeast p24 isoform, yp24A, is knocked out in a strain defective for vesicle fusion, a dramatic reduction in the accumulation of transport vesicles is observed. Together, these results indicate a role for this protein family in the budding of coatamer-coated and other species of coated vesicles.

Defining the invasive phenotype of proximal gastric cancer cells.

BACKGROUND: Adenocarcinoma of the proximal stomach is now the most rapidly rising cancer among men in the United States. The development of metastases is the major cause of morbidity and mortality for this aggressive disease. The mechanisms by which tumor cells invade the basement membrane are unknown for this disease. We have identified and established 5 invasive and noninvasive adenocarcinoma cell lines arising from the proximal stomach, which can be used to examine the mechanisms involved in tumor cell invasion. METHODS: The expression of factors associated with tumor cell attachment, proteolysis, and inhibition of proteolysis was determined by reverse transcription of mRNA to cDNA and subsequent amplification by the polymerase chain reaction. In addition, cells were examined for morphologic changes by scanning electron microscopy. RESULTS: Invasive proximal gastric cancer cells express the 72-kD form of collagenase type IV, whereas the noninvasive cells do not. Other factors examined (including the laminin receptor, cathepsin B, cathepsin L, urokinase-type plasminogen activator, and tissue inhibitor metalloproteinases) are expressed by both invasive and noninvasive gastric cancer cells, whereas collagenase type IV 92-kD form is not expressed by any of the cells examined. In addition, scanning electron microscopy revealed that all the invasive cell lines exhibit long cytoplasmic extensions. The noninvasive cells express short cytoplasmic projections and are rounder than the invasive proximal gastric cancer cell lines. CONCLUSIONS: There are distinct phenotypic differences between invasive and noninvasive proximal gastric cancer cell lines both at the level of expression of mRNA for collagenase Type IV 72-kD and at the level of scanning electron microscopy with the expression of cytoplasmic projections. Clinical outcome may be associated with these phenotypic differences.

Scanning electron microscopy of human leukocytes.

Extensive application of scanning electron microscopy (SEM) to the study of normal as well as of pathological human leukocytes has been pursued over the last ten years. Initial efforts to recognize, under the SEM, the two main subpopulations of circulating lymphocytes have met with limited success. However, a critical evaluation of the cell preparatory methods has emerged from the initial difficulties. Highly reproducible SEM images of leukocytes can now be obtained, and their significance correlated with immunological and/or cytochemical parameters. This resulted from the development of highly specific techniques for the immuno-labeling of cell surfaces, and from the development of SEM cytochemistry in the backscattered electron imaging (BEI) mode. A basic procedure for the SEI imaging of separated peripheral blood leukocytes (PBL's) is critically presented with the hope of facilitating significant future applications of SEM in diagnostic clinical hematology.

Backscattered electron imaging of human leukocytes.

Methods of backscattered electron imaging (BEI) have been applied to the study of normal and malignant human leukocytes. Four different elements, principally of high atomic number, have been used: iron, silver, lead and osmium. Iron, in the form of iron carbonyl, has been found to be an excellent marker for the phagocytic activity of granulocytes and monocytes. Silver staining permitted a clear observation of the shape of the nuclei and of the nucleo-cytoplasmic ratio and made the identification of polymorphonuclear cells and of certain normoblasts possible. Lead phosphate precipitates, deposited as a result of a classic acid phosphatase reaction, were detected in several cases of myelocytic and monocytic leukemias. Precipitates of osmium/diaminobenzidine complex on peroxidase-containing granules permitted the unambiguous identification of several classes of myeloid precursors. In all these techniques, the surface morphology of the cells was adequately preserved and could be correlated with the presence and/or the distribution of the various markers.

Labeling of lymphocyte surface immunoglobulins with T4 as marker for scanning electron microscopy.

The technique originally described by Kumon et al. for the labeling of cell surface antigens under the SEM has been adapted to the study of surface immunoglobulins on human lymphocytes. Briefly, the peripheral blood lymphocytes are: 1) separated in a Ficoll Hypaque gradient, 2) rinsed with culture medium, 3) allowed to attach to plastic coverslips, 4) prefixed with 1% glutaraldehyde for 10 minutes, 5) incubated with goat anti-human immunoglobulins for 30 minutes at 24 degrees C, 6) rinsed, 7) incubated with a rabbit anti-goat IgG coupled with purified bacteriophage T4, 8) rinsed, 9) postfixed in glutaraldehyde for several hours, and 10) dried from ethanol at -75 degrees C and under 10(-2) Torr. Results from normal human lymphocytes and cells from cases of Chronic Lymphoblastic Leukemia (CLL) indicate that the method makes it possible to recognize cells with surface immunoglobulins and permits some correlation between the surface architecture of the cells and the presence of surface Ig. It also permits the study under the SEM of T-derived lymphocytes and of null cells, the unlabeled cell population, without resorting to techniques for lymphocyte subclass separation which may alter surface morphology.

Massive lymphadenopathy mimicking lymphoma in leukemic reticuloendotheliosis.

Leukemic reticuloendotheliosis is increasingly noted to have a spectrum of laboratory findings suggestive of both lymphocytes and monocytes. However, previous reports have not noted a clinical presentation which may be confused with lymphoma. This report documents a case of leukemic reticuloendotheliosis in a 29 year old man with clinical findings of diffuse lymphadenopathy, organomegaly and cutaneous involvement. As cytotoxic agents may be dysfunctional in leukemic reticuloendotheliosis, the ability to distinguish between the disorder and a lymphomatous process may be critical to the patient's management. Both morphologic examination of the "hairy-cell" and cytochemistry may not give an unequivocal differentiation between these two diseases. However, functional studies of the neoplastic cell, such as cell-marker analysis, phagocytic function and ultrastructural morphology, can define by noninvasive methods the correct diagnosis in the atypical presentation of leukemic reticuloendotheliosis.

Mitotic spindles of Drosophila melanogaster: a phase-contrast and scanning electron-microscope study.

Mitotic spindles have been isolated from the blastema stage of Drosophila melanogaster embryos using modified tubulin-polymerizing medium. 'Clean' spindles, relatively free of contaminating cytoplasmic material, are obtained. Under phase contrast, mitotic stages appear remarkably similar to those seen in situ, as reported in early literature. This preservation of morphological integrity, coupled with relative structural simplicity due to low chromosome number (2n = 8), makes these spindles ideal subjects for study. Use of the scanning electron microscope provides excellent visulization of their general structural organization, changes in whole spindle structure during the course of mitosis, and higher resolution viewing of surface detail than is permitted with light microscopy.

A scanning electron microscopy and immunological study of 84 cases of lymphocytic leukaemia and related lymphoproliferative disorders.

The surface features of cells from 84 cases of lymphocytic leukaemia, and related lymphoproliferative disorders are described as seen by scanning electron microscopy (SEM). Most of the 46 cases of CLL were shown to be B-derived, but rare cases of mixed B and T cell leukaemia and leukaemia with cells bearing both B and T markers were also encountered. Despite the existence of a spectrum of cell surface morphology, it was possible in many cases to identify a dominant cell type. Cells from cases of B derived malignancies were most frequently of the 'predominantly villous' type while a smaller proportion of cases were of the predominantly 'smoother' or 'mixed villous and smooth' type. Variations in surface morphology also occurred with progression of the disease. In most cases of acute lymphoblastic leukaemia (ALL) 'smoother' cells predominated. However, more cases of ALL and T derived leukaemia need to be examined before definite conclusions can be drawn concerning the surface of these cell types. This study also illustrates the importance of examining large numbers of cases of leukaemia, before conclusions are drawn concerning their surface features and indicates that SEM cannot consistently distinguish between leukaemic B and T cells. It will be of interest to determine whether the surface architecture of the leukaemic cell is related to the degree of cell differentiation and eventual prognosis in these cases.

A scanning electron microscopic study of 34 cases of acute granulocytic, myelomonocytic, monoblastic and histiocytic leukemia.

This report describes the surface architecture of leukemic cells, as seen by scanning electron microscopy in 34 patients with acute nonlymphoblastic leukemia. Six patients with myeloblastic, 4 with promyelocytic, 10 with myelomonocytic, 8 with monocytic, 4 with histiocytic and 2 with undifferentiated leukemia were studied. Under the scanning electron microscope most leukemia histiocytes and monocytes appeared similar and were characterized by the presence of large, well developed broad-based ruffled membranes or prominent raised ridge-like profiles, resembling ithis respect normal monocytes. Most cells from patients with acute promyelocytic or myeloblastic leukemia exhibited narrower ridge-like profiles whereas some showed ruffles or microvilli. Patients with myelomonocytic leukemia showed mixed populations of cells with ridge-like profiles and ruffled membranes whereas cells from two patients with undifferentiated leukemia had smooth surfaces, similar to those encountered in cells from patients with acute lymphoblastic leukemia. It appears that nonlymphoblastic and lymphoblastic leukemia cells (particularly histiocytes and monocytes) can frequently be distinquished on the basis of their surface architecture. The surface features of leukemic histiocytes and monocytes are similar, suggesting that they may belong to the same cell series. The monocytes seem to have characteristic surface features recognizable with the scanning electron microscope and differ from most cells from patients with acute granulocytic leukemia. Although overlap of surface features and misidentification can occur, scanning electron microscopy is a useful adjunct to other modes of microscopy in the study and diagnosis of acute leukemia.

Surface morphology of murine B and T lymphocytes: A comparative study by scanning electron microscopy.

A variety of murine lymphocytes of known B or T derivation obtained from different lymphoid organs were prepared for scanning electron microscopy (SEM) by the critical point drying method after collecting the cells by aspiration onto silver membranes. Comparison of SEM appearances of cells prepared by this technique and serological classification according to surface antigens showed that most T cells had smooth surfaces with few microvilli, while many B lymphocytes were moderately to markedly villous. Further evidence for the above correlation was obtained by examining thymic cells and enriched B or T cell populations. Thymic cell suspensions containing less than 5% B cells showed over 80% generally smooth cells by SEM. Enriched T cell populations, obtained by mass cytolysis of lymph node preparations with anti-Ia or anti-Ig sera or by purification through nylon fiber columns, contained over 85% T cells, and more than 75% of them were of the smooth cell type. A similar correlation was noted for enriched B cell populations obtained by cytolysis of lymph node cells with anti-Thy-1 serum, and by lysis of EAC-rosettes. Over 90% of these cells were identified as B cells by immunologic methods and approximately 75% had moderate to markedly villous surfaces. The 15% difference can be accounted for by the existence of a subpopulation of smooth B cells. Direct observation of EAC-rosettes confirmed that most B cells had moderate to large numbers of surface microvilli and that less than 10% were smooth. It is possible that some of the smooth cells seen in enriched B cell populations may represent precursors or B lymphocytes at different stages of differentiation. These results indicate that murine T and B lymphocytes, like their human counterparts, can be recognized in many cases under the SEM on the basis of their surface morphology. Smoother B and more villous T cells are difficult to classify by SEM without parallel immunologic identification.

Scanning electron microscopy of human lymphocyte-sheep erythrocyte rosettes.

Human lymphocytes of known B or T derivation were examined by scanning electron microscopy (SEM) before and after rosetting with SRBC. After collection of the cells onto silver membranes the samples were prepared for SEM by the critical point drying method. Sheep RBC frequently underwent sphero-echinocyte transformation and multiple projections extended from their surfaces. This was readily noticeable after storage of SRBC in the cold and washing in Hanks, but more prominent after rosetting. These erythrocyte surface alterations were less apparent when freshly withdrawn cells were used. Spontaneous sheep erythrocyte rosettes (E-R), a marker for human T lymphocytes, were prepared with normal peripheral blood lymphocytes (PBL), thymic cells, and cultured T cells. EAC-rosettes (EAC-R), used to identify B lymphocytes with complement receptors, were prepared with normal PBL and cultured B cells. The majority of rosetting T lymphocytes had generally smooth surfaces while about 20% had an intermediate number of microvilli and 15% were more villous and indistinguishable from villous B cells. Studies of rosetting thymocytes and cultured T cells however indicated that the surface of some T cells alters on rosetting, becoming more villous and thus account for the higher numbers of villous T cells seen in E-rosettes. Point to point contact sites between SRBC and T lymphocytes were more frequent than broad zones of attachment. The majority of rosetting B lymphocytes had multiple microvilli, about 25% had a moderate number of microvilli and less than 10% had smooth surfaces similar to those of most T cells. Areas of contact between EAC and B lymphocytes were frequently broad zones of attachment. The study confirms that in many cases B and T lymphocytes can be distinguished by their surface architecture as seen under the SEM; however, about 20% of rosetting B and T cells have similar surfaces with intermediate numbers of surface microvilli and cannot be distinguished by SEM without parallel immunologic identification.

Identification of human B and T lymphocytes by scanning electron microscopy.

In this study a variety of human lymphocytes of known B or T cell type, obtained from multiple sources, were prepared for scanning electron microscopy (SEM) by the critical point drying method. Distinction between normal B and T lymphocytes was relatively easy in most instances, on the basis of their surface architecture. Using immunological methods, between 20 and 30% of normal peripheral blood lymphocytes (PBL) were identified as B cells and from 69 to 82% as T cells. SEM results showed that 20% of the PBL had a complex villous surface and approximately 80% of cells were smaller and had a relatively smooth surface. Comparison of the above data and enrichment of B cells from PBL, by centrifugation after T cell rosettes had formed, indicated that the "villous" cells were B lymphocytes and the "relatively smooth" cells were T lymphocytes. T cells obtained from two human thymuses were also of the generally smooth cell type. Further evidence for the distinction of B and T lymphocytes, on the basis of surface morphology, was obtained from the examination of cultured lymphoid cell lines of known B or T cell derivation. Cells from cases of chronic lymphocytic leukemia also provided support for the above interpretations. Five of six untreated cases were clearly of B cell type by immunologic and SEM criteria. One unusual case showed the presence of T and B lymphocytes in almost equal numbers by SEM and a mixture of B and T cells by immunologic markers. An additional case that had received chemotherapy showed numerous atypical cells that were difficult to classify by SEM. Detailed examination of the smoother T cells showed that at least half of them had a moderate number of surface digitations and a small proportion had an intermediate surface morphology with a relatively large number of surface digitations. The latter presented difficulties in classification and may correspond to different stages of differentiation and represent subpopulations of lymphocytes. The distinction between human B and T lymphocytes on the basis of their surface architecture can be made by SEM of critical point dried samples, with relative ease in most but not all instances. The effects of stimulation, cell cycle, differentiation, intercellular contact, and density of cell population, on the surface architecture of lymphoid cells, remain to be determined.