Selma Candelária Genari

Cytotoxicity of tamoxifen in normal and tumoral cell lines and its ability to induce cellular transformation in vitro

Tamoxifen (TAM) is a non‐steroidal anti‐estrogen used to treat patients with estrogen receptor‐positive breast cancer and as a chemopreventive agent against breast cancer in high risk pre‐ and post‐menopausal women. However, recent studies have shown that tamoxifen causes endometrial and hepatic cancer. In this study, we examined the effects of tamoxifen (5, 10, 25 and 50 μM) on the growth and proliferation of nine tumoral cell lines (UACC62, MCF‐7, NCI‐460, K‐562, OVCAR‐03, PC‐03, HT‐29, 786‐0, NCI‐ADR) and non‐tumoral cell lines (3T3, V79, MDCK, VERO). Chinese hamster lung fibroblasts (V79) were the most sensitive lineage to tamoxifen, with 21.6% of the cells showing apoptosis at 50 μM TAM. Microscopic analysis showed that, the cellular transformation caused by TAM in V79 cells was similar to that seen with 7,12‐dimethylbenz(a)anthracene, thus indicating the carcinogenicity of TAM.

Morphological and growth alterations in Vero cells transformed by cisplatin

Cisplatin is an antineoplastic agent used to treat solid tumours, such as ovarian, testicular and bladder tumours. However, studies in vitro and in vivo have shown that cisplatin is mutagenic, genotoxic and tumorigenic in other tissues and organs. In this work, we examined the effect of cisplatin on Vero cells, a fibroblast‐like cell line. The morphological characteristics were investigated using phase contrast microscopy, scanning electron microscopy and the actin cytoskeleton was labelled with fluorescein isothiocyanate‐phalloidin. Cell proliferation was assessed based on the growth curve. Cultured Vero cells treated with cisplatin showed behavioural and morphological alterations associated with cellular transformation. The transformed cells grew in multilayers and formed cellular aggregates. The proliferation and morphological characteristics of the transformed cells were very different from those of control ones. Since transformed Vero cells showed several characteristics related to neoplastic growth, these cells could be a useful model for studying tumour cells in vitro.

ALTERATIONS IN THE GROWTH AND ADHESION PATTERN OF VERO CELLS INDUCED BY NUTRITIONAL STRESS CONDITIONS

The pattern of growth, adhesion and protein synthesis in Vero cells submitted to nutritional stress conditions was investigated. The control cells presented a characteristic pattern, with monolayer growth, while the stressed cells presented multilayered growth, with aggregate or spheroid formation which detached on the flask surface and continued their growth in another region. In the soft agar assay, with reduced amount of nutrients, only the stressed cells presented growth, indicating physical and nutritional independence. A 44‐kDa protein was observed in stressed cells and was absent in non‐stressed cells. The adhesion index and fibronectin synthesis and distribution were altered in stressed cells. After confluence, control cells presented fibronectin accumulation in lateral cell—cell contact regions, while this fibronectin accumulation pattern was not observed in stressed cells. These alterations may be responsible for the multilayered growth and decreased adhesion index observed in stressed cells which were transformed by nutritional stress conditions.

Bovine osteoblasts cultured on polyanionic collagen scaffolds: An ultrastructural and immunocytochemical study †

Collagen is the most abundant protein in the body and is also the most important component of the extracellular matrix. Collagen has several advantages as a biomaterial such as lack of toxicity, biocompatibility, biodegradability, and easy reabsorption. In this study, we examined bovine osteoblasts cultured on native or anionic collagen scaffolds prepared from bovine pericardium after selective hydrolysis of glutamine and asparagine side chain amides for periods from 24 (BP24) and 48 h (BP48). The cells were cultured in control and mineralization medium at 37 °C in the presence of 5% CO(2). Transmission and scanning electron microscopy, energy dispersive spectroscopy, and an immunocytochemical marker were used for analysis. Cells with an irregular morphology forming a confluent multilayer were observed on matrices kept in control medium. Most of these cells presented a polygonal or elongated flattened morphology. Several spherical deposits of calcium crystal associated with phosphorus were observed on the native and BP48 matrices. Similar results were observed in samples kept in control medium except with lower calcium/phosphorus ratio. Vesicles actively expelled from the cell membrane were also seen (do this vesicles corresponds to calcium/phosphorus deposits). Osteocalcin was clearly visible on matrices kept in mineralization medium and was more expression on the surface of BP48 matrices. The results showed that anionic collagen is able to support osteoblastic differentiation, regardless of the medium used. Finally, the BP48 matrix promoted better osteoblast differentiation than the native matrix.

Technologies Applied to Stimulate Bone Regeneration

Arnaldo Rodrigues Santos Jr.1, Christiane Bertachini Lombello2 and Selma Candelaria Genari3 1Centro de Ciencias Naturais e Humanas (CCNH), Universidade Federal do ABC, Santo Andre, SP; 2Centro de Engenharia e Ciencias Sociais Aplicadas (CECS), Universidade Federal do ABC, Santo Andre, SP; 3Centro Estadual de Educacao Tecnologica Paula Souza, Faculdade de Tecnologia de Bauru (FATEC), Bauru, SP; Brazil

Ultrastructural Characterization of the New NG97ht Human-Derived Glioma Cell Line Using Two Different Electron Microscopy Technical Procedures

On the basis of transmission electron microscopy observations in tumor cell lines, oncologists have made innumerous diagnostic and therapeutical progresses. Following this path, the UNICAMP immunopathologies laboratory established the NG97 cell line derived from a human astrocytoma grade III, which when injected to the athymic nude mouse flank developed a grade IV astrocytoma. In this study, we focused on ultrastructural characterization of the NG97 cells after being recovered from xenotransplant (NG97ht). These cells in culture were assayed by two different electron microscopy procedures to characterize ultrastructures related to grade IV astrocytomas and to observe their structures through cell subcultivation. Additionally, comparative morphological descriptions of different cell passages in these technical procedures could be a useful tool for improving electron microscopy cell lineage protocols. Results from many cell passage observations showed ultrastructural similarities, which suggest malignant and glioblastoma phenotypes. In the first procedure, NG97ht cells were harvested and then incorporated into agarose before subjecting them to electron microscopy protocols, whereas in the second one, monolayer cells grew first on cover slides. Comparison among protocols revealed that organelles, cytoplasmatic extensions, spatial conformation of filopodia, and cell attachment to substrate were more preserved in the second procedure. Furthermore, in this latter procedure, a unique ellipsoidal structure was observed, which was already described when dealing with gliosarcoma cell line elsewhere. Therefore, these analyses demonstrated a morphological characterization of a new NG97ht cell line using electron transmission microscopy. Moreover, it has been shown that the second procedure provides more detailed information compared with the first. Microsc. Res. Tech, 2009.

Evaluation of Cell Growth Characteristics on Chitosan-Alginate Membranes to Assess Their Potential Application on Highly Exuding Skin Lesions and In Vivo Evaluation in Wounded Cat

Chitosan and alginate are non-toxic, biocompatible and biodegradable polysaccharides, used in skin healing. High biocompatibility, ease of production and low cost have increased the interest in the use of both compounds in medical and pharmaceutical sectors, as components of wound dressings. The purpose of this work was to access the use of chitosan and chitosan-alginate membranes for highly exudating wounds, as the ones normally observed in cats, employing Vero cell cul- ture as an initial evaluation step. The achieved results show that the chitosan-alginate membranes stimulated cell proliferation, but not cell adhesion, whereas improving tridimentional growth with the formation of aggregates. Cell concentration both at 24 and 48 h were similar to those observed in the control samples, however, at the 72-h period, the average number of cells was lower than those in the controls. The in vivo performance evaluation of the membranes indicated that they were effective on protecting the wound from external microbial contamination, also stimulating tissue recovery. The lesion was totally healed after 30 days.

Light microscope observation of circulating human lymphocytes cultured in vitro

Os linfocitos sao celulas importantes do sistema imune e tem sido largamente utilizados em estudos morfologicos. Entretanto, a literatura sobre tecnicas de preparacao dessas celulas e escassa e antiga, especialmente para linfocitos cultivados in vitro. Portanto, o objetivo desse estudo foi relatar com detalhes as tecnicas de isolamento e microscopia de luz de linfocitos mantidos em cultura. Amostras de sangue foram obtidas por puncao venosa e centrifugadas em gradiente de densidade de Percoll, para separar os leucocitos. Os linfocitos foram mantidos em frascos de cultura de 25 cm3 a 37oC. Apos a cultura, as celulas foram fixadas e coradas de acordo com a metodologia utilizada para esfregacos sanguineos. Nossos resultados mostraram que nem todos os fixadores e corantes utilizados para esfregacos sanguineos sao uma boa escolha para linfocitos cultivados in vitro.