Volume 44 (1998) No. 2
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- S. GABERŠÈEK1, D. ŠTIBLAR-MARTINÈIÈ2, M. KALIŠNIK2
- L. HERINGOVÁ, R. JELÍNEK
- The world-wide and long-lasting stagnation in general incidence of inborn defects does not agree with the increasing impact of the environment. However, inborn defects alone are definitely a poor indicator of the reproductive risk being accompanied by much more frequent embryotoxicity manifestations - death of the conceptus and growth retardation. Dose-dependent transformations of the embryotoxicity manifestations may explain both the stagnation in the incidence of inborn defects and the observed shifts in malformation spectra. An attempt was made to design a reliable experimental model to produce individual transformations resulting from the specific dose-response relationships inherited in teratogenesis. A population of chick embryos in different stages of development was exposed to increasing doses of cyclophosphamide, a model teratogen. All types of the transformations were recorded. Prenatal extinction of defective embryos, transition of single moderate defects to more severe and multiple malformations, as well as the changes in the incidence of certain types of defects exhibit a clear-cut positive dose dependence. Introducing this experimental system we will be able to study mechanisms underlying the particular transformations.
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- Derived from Human Keratinocytes and Dried Porcine Dermis
- E. MATOUŠKOVÁ1, I. McKAY2, C. POVÝŠIL3, R. KÖNIGOVÁ4, A. CHALOUPKOVÁ1, P. VESELÝ1
- A number of skin models have been developed, but a simple method for rapidly producing large quantities of differentiated epidermis has been missing. We show the differentiated phenotype of human keratinocytes in organotypic culture arising in vitro by air-exposure of keratinocytes cultured with feeders on dried pig dermis. Keratinocytes were seeded at low density on the dermis covered with irradiated NIH-3T3 feeder cells and after reaching confluence lifted to the air-medium interface. A well differentiated epidermis with distinct basal, spinous, granular and stratum corneum layers was formed within 1 week. In this way, 100 cm2 of the differentiated recombined human/pig skin (D-RHPS) can be obtained from 106 secondary keratinocytes in 15 days. The entire keratinocyte life cycle takes place on the dermal substrate - from single cells to stratified epidermis. The differentiation was characterized using a panel of monoclonal antibodies. Similarly as in the normal skin, keratin 14 was expressed in all cell layers, keratin 10 in suprabasal layers, Ð1 integrin and epitopes to antibody LH8 in the basal layer, involucrin and transglutaminase in the granular and horny layer of the epidermis. Keratins 16 and 7/17, which are absent in the normal epidermis, but present suprabasally in the psoriatic one, were expressed strongly in all suprabasal layers and in a subpopulation of basal cells. The keratinocytes can be combined with two other cell types cultured either on the dermal side of the dermis and/or on the bottom of the dish. It appears that this simple skin model can be used in studies of epithelial/mesenchymal interactions and interactions between epidermal cells and infectious agents. It may be particularly useful for the study of human papilloma viruses.@LH 6
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Short Communication- Clonal Expansion of Epithelial Cells from Primary Human Breast Carcinoma with 3T3 Feeder Layer Technique
- E. MATOUŠKOVÁ1, D. DUDORKINOVÁ2, L'. PAVLÍKOVÁ1, C. POVÝŠIL2, P. VESELÝ1
- 3T3 feeder layer technique provided support for colony growth and serial propagation of two apparently single epithelial cells isolated from a peroperative biopsy of a primary ductal breast carcinoma. The total culture lifetime was estimated to be more than 30 doublings, 21 of which took place during the primary culture. The two cells were the only survivors of two-week exposure to stressing conditions that resembled the microenvironment in a tumour (low pH, depleted nutrition and accumulation of metabolic waste). The epithelial character of the cells was proved by positive immunostaining for keratins 7/17. The majority of growing cells did not express keratin 19. Only quiescent cells in some colonies, which appeared to reach a more advanced stage of differentiation, expressed keratin 19. These features correspond with the characteristics of mammary luminal cells which in vivo undergo differentiation from the stem K19- to secretory K19+ cells. The luminal cells are supposed to be the target of malignant transformation in the mammary gland. The described technique opens a regular way for the in vitro clonal growth of individual primary cells from breast tumours. Such an approach can improve our understanding of the biology of breast cancer cell populations and also simplify the predictive chemosensitivity assay on breast cancer cells from individual patients.
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The present work is based on the results of in vivo experiments on rats, which had shown that hypercalcemia had led to morphological and biochemical hyperfunction of thyroid follicular cells. The regulation of the activity of follicular cells should directly, or indirectly via paracrine action of serotonin secreted from parafollicular cells, depend on the presence of calcium ions. The effect of calcium was studied on a cell line of rat follicular cells FRTL-5 (Fischer Rat Thyroid cells in Low serum) using three methods: measuring the quantity of produced cAMP (cyclic adenosine 3',5'-monophosphate), measuring [3H]thymidine incorporation into cell DNA and transmission electron microscopy. Results show that calcium has no effect on cAMP production. Calcium at 1.3 mM, 3 mM, 10 mM, 20 mM and 30 mM concentrations increases [3H]thymidine incorporation into cell DNA when compared with controls without calcium. Calcium at the concentration of 30 mM has no effect on FRTL-5 cell morphology. TSH (thyrotropin) stimulates follicular cells; at higher extracellular concentrations (3 mM, 10 mM, 20 mM, 30 mM), calcium diminishes its effect, presumably by activation of a cAMP phosphodiesterase which disintegrates cAMP and/or by inhibition of adenyl cyclase.
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