Tsukasa Tanii

Cell cycle analysis of human dermal fibroblasts cultured on or in hydrated type I collagen lattices

SummaryThe proliferation and cell cycle phase composition of human dermal fibroblasts cultured on or in type I collagen lattices (reconstituted dermis model) were examined. On collagen lattices, as compared with conventional cultures on plastic dishes, the proliferation of human dermal fibroblasts was suppressed, being arrested at about one-half the saturation density after 10 days of culture. In collagen lattices, proliferation was further suppressed, being nearly arrested within 4–7 days of culture. Cells were analyzed for cell cycle phases by two-color flow cytometry using DNA staining and S phase cell staining with FITC-conjugated antibromodeoxyuridine antibody. After 5 days of culture, the number of S phase cells on collagen lattices was 49.3% of that on plastic dishes, with an increase in G0G1 phase cells of 79.8%. In collagen lattices, the number of S phase cells was very small (4.3% of all cells), and most of the cells accumulated in G0G1 phase. These findings suggest that the cell cycle of fibroblasts is arrested at G0G1 phase by their interaction with collagen. On the basis of these results, the reconstituted dermis model using collagen lattice is considered to be analogous to the dermis in vivo with respect to cell growth and cell cycle phase composition.

Parallel Arrangement, Growth Inhibition and Cell Cycle Phase Analysis of Human Dermal Fibroblasts Cultured in Collagen Lattice

Human dermal fibroblasts were cultured in a hydrated type I collagen lattice. When collagen fibers were arranged in one direction, fibroblasts were arranged in the same direction. Cell proliferation was markedly suppressed in the collagen lattice as compared with that on plastic, with growth being arrested after day 5. No differences in proliferation were observed between aligned cells and randomly oriented cells.

A Variant of Encephalomeningocele: Heterotopic Brain Tissue on the Scalp

A 2-year-old girl having a bald nodule on the scalp is reported. The tumor was circular, hemispherically elevated and had a dull reddish color. Roentgenologic studies revealed a defect of the underlying skull bone, and spina bifida occulta on the first and second sacral vertebra were found. Craniotomy was carried out. The tumor extended into the falx cerebri and reached to the tentorium cerebelli, and was not connected with the cerebrum. Histological examination showed insular brain tissue containing glial cells in the lower dermis. Around this tissue, there seemed to be vascular leptomeninges with numerous melanocytes and thickened fibrous elements, probably dura. From these findings, this case was diagnosed as a variant of encephalomeningocele, and as melanocytes physiologically exist on the leptomeninges, melanocytes in an encephalomeningocele may diagnostically identify the leptomeninges.

Effects of Human Recombinant Tumor Necrosis Factor‐α (TNF‐α) on the Proliferative Potential of Human Keratinocytes Cultured in Serum‐free Medium

The effects of human recombinant tumor necrosis factor‐α (TNF‐α) on human keratinocytes cultured in a serum‐free medium were investigated. TNF‐α markedly suppressed cell growth. The growth‐inhibitory effect was reversible and cytostatic at a concentration of 1–5 U/ml, but appeared to be irreversible and cytocidal at 10 U/ml. The growth suppressive effect was more marked when TNF‐α was added in the late growth phase or preconfluent phase than when it was added in early or mid‐growth phases. No effects of TNF‐α on cell adhesion to the substrate were observed. These results indicate that TNF‐α is a very potent anti‐proliferative agent for human keratinocytes.

Correlation of Contractility and Proliferative Potential with the Extent of Differentiation in Mouse Fibroblastic Cell Lines Cultured in Collagen Lattices

Four types of fibroblastic cell lines at various stage of differentiation, which had been derived from syngeneic mice, were cultured in collagen lattices (reconstituted dermis model). Lattice contraction, growth in the lattice, and cell morphology were compared. The following cell lines were used: [I] precrisis cells within several subcultures derived from the skin of Balb/c mice, [II] an established normal cell line derived from syngeneic mice (Balb/3T3 clone A31), and [III] two transformed lines (Balb/3T12‐3, 3T3‐B‐SV40) originating from [II]. The cells adopted a bipolar spindle form in the collagen lattice. Lattice contraction was the most marked with cell type [I] followed in order by [II] and [III]. Relative growth in the lattice occurred in the reverse order (III>II>I). These findings suggested a correlation between lattice contraction and growth in the lattice and also between the extent of differentiation and lattice contraction.

Effects of Ryo-Kan-Kyomi-Sin-Ge-Nin-To Extract on Degranulation of and Histamine Release from Rat Mast Cells

The effects of Ryo-kan-kyomi-sin-ge-nin-to (RKSG)) extract, a medicinal agent traditionally used in China and Japan for treatment of asthma, on the degranulation of and histamine release from rat mast cells were studied. At a concentration of 5 mg/ml RKSG, degranulation of mast cells stimulated either by antigen (DNP-Ascaris) or compound 48/80 was markedly suppressed. At a concentration of 1-5 mg/ml RKSG, histamine release from mast cells due to application of either antigen or compound 48/80 was inhibited in a dose-dependent fashion. These results suggest that RKSG may be useful for the treatment of type I allergy-related diseases.

Proliferative Potential of Murine Melanoma Cells Cultured in or on Collagen Gel

As the first step in developing an in vitro model of melanoma cells infiltrating the dermis, B16 murine melanoma cells were cultured on and in type I collagen gels. Under these conditions, the melanoma cell adopted an elongated or dendritic form. Cell proliferation was suppressed in the culture system using the collagen gel as compared with the conventional monolayer culture on plastic. Microcinematographically, this suppression was found to be due to an extension of the cell cycle time of each individual cell.

Secondary T-cell lymphoma presenting as a giant ulcer

Secondary cutaneous T‐cell lymphoma may present with various types of skin lesions, hut rarely produces ulceration in contrast to primary cutaneous T‐cell lymphoma. We report a case of malignant lymphoma of the tonsil that recurred as a giant cutaneous ulcer on the back of a 45‐year‐old man, Biopsy of the ulcer revealed a malignant lymphoma of diffuse, mixed‐cell type. Surface marker analysis of the tumour cells showed suppressor/cytotoxic T‐cell characteristics. The skin lesions were treated by electron beam therapy.

Effect of tretinoin on collagen gel contraction induced by mouse 3T3 fibroblasts

Balb/3T3 fibroblasts were cultured in type I collagen gel and the effects of tretinoin (all-trans-retinoic acid) were examined on cell growth and the gel contraction produced by cells. Cell proliferation was suppressed and the degree of gel contraction was enhanced by the addition of 10(-7) and 10(-6) M tretinoin. Growth and gel contractility of transformed cells derived from the Balb/3T3 cells were not influenced by this agent. Addition of 12-O-tetradecanoylphorbol ester, which is known to antagonize tretinoin in several biological processes, enhanced gel contraction synergistically with tretinoin. These results suggest that tretinoin influences cell-to-collagen interactions.

Infiltration of melanoma cells into the type I collagen gel.

Melanoma cells were cultured on type I collagen gel, and the infiltration of those cells into the gel was observed. B16 murine melanoma cells initially adopted a spherical form on the gel, but they assumed a dendritic form after infiltration into the interior. The degree of infiltration increased very rapidly and was time‐dependent. No correlations between the growth rate or melanogenic activity and infiltrative potential were observed. When Syrian hamster and human melanoma cell lines were cultured, the degrees of infiltration varied. This culture system using collagen gel is considered to be a useful in vitro model of tumor cell invasion.