Dennis L. Way

CHARACTERIZATION OF A NEW HUMAN ENDOMETRIAL CARCINOMA (RL95-2) ESTABLISHED IN TISSUE CULTURE

SummaryA new human endometrial cell line, RL95-2, derived from a Grade 2 moderately differentiated adenosquamous carcinoma of the endometrium has been passaged successfully in cell culture for more than 2 yr. The cells are characteristically epithelioid with well-defined junctional complexes, tonofilaments, filopodialike extensions, and surface microvilli. Nuclei are large, irregular, and invaginated frequently with multiple, prominent, lamellar nucleoli. The cells have a log phase doubling time of 22 to 34 h followed by continued growth at a reduced rate with no apparent plateau phase. They exhibit a strong tendency for piling up as well as for the formation of glandlike dome structures. Karyotypically the line is trisomic 8 (47,XX,+8) and has an 8% frequency of polyploidization. Both cytoplasmic and nuclear estrogen receptors are present. Antihuman α-keratin characterizes the cell line as epithelial, nonstromal. The RL95-2 cell line may provide a useful in vitro system for the investigation of the endocrine regulation of endometrial neoplasia.

Lymphangiogenesis: Mechanisms, significance and clinical implications

Great attention has been directed toward understanding angiogenesis over the past several decades since this phenomenon was reproduced in vitro in endothelial cell and mixed vascular tissue cultures (Folkman and Hauden-schild, 1980; Folkman, 1995). The focus, however, has been almost entirely on blood vessel growth, or what we have termed “hemangiogenesis” (M. Witte and Witte, 1987 c). Yet a vast interstitial fluid circulation suffuses the tissues, bathes the parenchymal cells and interconnects with the blood vasculature via the lymphatic vasculature (Fig. 1). This arc of the circulatory system on the dark side of the blood-tissue interface and its growth (“lymphangiogenesis”) has received scant attention even though lymphatic (re)generation is both vigorous and essential, and disorders of lymphatic dynamics are common, often disfiguring, and occasionally life- and limb-threatening.

The restricted cellular host range of human herpesvirus 8

DesignA selection of primary and transformed cell types were evaluated for their susceptibility to infection with human herpesvirus 8 (HHV-8)/Kaposis sarcoma- associated herpesvirus. MethodsSources of HHV-8 included Kaposis sarcoma lesion punch biopsies that were either cocultured directly with target cells or that were first cocultured with human lymphocytes to derive HHV-8-containing fluids that were inoculated onto target cells. HHV-8 was also obtained from primary effusion lymphoma-derived cell lines. Techniques to detect infection included the PCR, immunofluorescence assays and in situ hybridization. ResultsSusceptible cells included human umbilical cord blood mononuclear cells (UCMC), adult CD19 B cells, macrophages and certain endothelial cells of human and animal origin, including some that are transformed with human papilloma virus type 16 E6 and E7 genes. The infection of lymphocytes did not yield established lymphoblastoid cell lines (LCL) and virus infection persisted for only 4–7 days. However, long-term HHV-8 infection of UCMC could be achieved by coinfection with Epstein–Barr virus. HHV-8 could also infect UCMC LCL recently derived by Epstein–Barr virus transformation, but long-established LCL could not be infected with HHV-8. ConclusionsThese data provide further biological evidence in cell culture for the limited cellular host range of HHV-8 to CD19 B cells, macrophages, and certain endothelial cells.

DNA ploidy, grade, and stage in prognosis of uterine cervical cancer

A retrospective study of 56 cases of uterine cervical squamous carcinoma evaluated DNA content, histological grade, and clinical stage as indicators of prognosis. Minimum survivor follow-up was 24 months. Following standard radiation therapy, there were 40 cures and 16 treatment failures. DNA content was measured by flow cytometry of pretreatment biopsies removed from paraffin. There were 18 diploid cases and 38 aneuploid (67.9%). Aneuploid cases included 6 with very high G2-M peaks (greater than or equal to 15% of the cell sample). DNA ploidy correlation with prognosis was not statistically significant. However, both grading by a multiple parameter method (P less than 0.0133) and staging (P less than 0.0064) were significant prognostic factors. Higher grade and stage correlated with treatment failure.

A NOVEL TYPE OF ADHERING JUNCTION IN AN EPITHELIOID TUMORIGENIC RAT CELL CULTURE LINE

Abstract Two major types of plaque-bearing adhering junctions are commonly distinguished: the actin microfilament-anchoring adhaerens junctions (AJs) and the desmosomes anchoring intermediate-sized filaments (IFs). Both types of junction usually possess the common plaque protein, plakoglobin, whereas the other plaque proteins and the transmembrane cadherins are mutually exclusive. For example, AJs contain E-, N-, or P-cadherin in combination with α- and β-catenin, vinculin and α-actinin, whereas in desmosomes, desmogleins and desmocollins are associated with desmoplakin and one or several of the plakophilins (PP1–3). Here we describe a novel type of adhering junction comprising proteins of both AJs and desmosomes and the tight junction (TJ) plaque protein, ZO-1, in a newly established, liver-derived tumorigenic rat cell line (RMEC-1). By immunofluorescence microscopy, cell-cell contacts are characterized by mostly continuous-appearing lines which are usually resolved by electron microscopy as extended arrays of closely spaced small plaque subunits. These plaque-covered regions are positive for plakoglobin, α- and β-catenin, the arm-repeat protein p120, vinculin, desmoplakin and protein ZO-1. They are positive for E-cadherin in cultures early on in passaging, but tend to turn negative for all known cadherins in densely grown cultures. On immunoblotting SDS-PAGE-separated proteins from dense-grown cell monolayers, “pan-cadherin” antibodies have reacted with a band at ∼140 kDa, identified as N-cadherin by peptide fingerprinting of the immunoprecipitated protein, which for reasons not yet clear is modified or masked in immunolocalization experiments. The exact histological derivation of RMEC-1 cells is not known. However, the observations of several endothelial markers and the fact that all cells are rich in IFs containing vimentin and/or desmin, while only subpopulations also reveal IFs containing CKs 8 and 18, is suggestive of a mesenchymal, probably endothelial origin. We discuss the molecular relationship of this novel type of extended junction with other types of adhering junctions.

AIDS, alcohol, endothelium, and immunity

Abstract Analogies are drawn between important unknowns in AIDS and alcohol research, related to underlying common pathogenetic mechanisms, immunodysregulation, cofactors, and prominent vascular manifestations. The central role of the blood and lymphatic vasculatures and specifically their endothelial lining in many facets of the immune response is reviewed. Evidence is presented that both alcohol and HIV (as well as other coinfecting viruses in AIDS) target and alter endothelial cells and the angiogenic process. These concepts are further illustrated by a serendipitous viral epidemic among rats on continuous long-term alcoholic and control nonalcoholic diets, where synergism between alcohol and virus appeared to underlie multiple vascular proliferative lesions in the liver. In AIDS and alcoholism/alcoholic liver disease (ALD), the prominent features of dysregulated angiogenesis point to the endothelium as a key player in pathogenesis of these seemingly disparate disorders and potentially in immunomodulation.

Effective stabilization of ethanol levels in multiple-well tissue culture plates.

Underestimation of ethanol (EtOH) volatility in vitro is a potential source of experimental error. EtOH (0-5% in culture medium) was added to 24- or 96-well tissue culture plates with standard low evaporation lids and incubated at 37 degrees C in humidified 7.5% CO2 and 92.5% air. After 72 hours, approximately 70% of the initial EtOH had disappeared from the aqueous phase of the plate (EtOH volatilization). EtOH concentrations gradually decreased in high-concentration wells (1-5%) and increased in low-concentration wells (0-0.1%) over time. This temporal redistribution of EtOH (EtOH diffusion) was detected after only 1 hour of incubation. Parafilm, Blenderm surgical tape, and ELISA plate-sealing tape barriers inconsistently or inadequately prevented EtOH volatilization and diffusion, but a newly designed plate-sealing clamp (PSC) apparatus inhibited this phenomenon. Rat hepatic sinusoidal endothelial cells cultured with the PSC apparatus maintained intact cell membranes for 72 hours and stable levels of monolayer permeability for at least 48 hours. By stabilizing in vitro EtOH concentrations, the PSC apparatus eliminates a potential source of major experimental error.