Kazuaki Takada

Immunohistochemical study of overexpression of fibroblast growth factor-1 (FGF-1), FGF-2, and FGF receptor-1 in human malignant salivary gland tumours.

Fibroblast growth factor‐1 (FGF‐1) and FGF‐2 are broad spectrum mitogens. The expression of FGF‐1, FGF‐2, and their receptor, FGF receptor‐1 (FGFR‐1), was examined in malignant salivary gland tumours and normal salivary glands, using immunohistochemical methods. In seven cases of adenoid cystic carcinoma (ACC), both duct‐like cells and modified myoepithelial cells were apparently immunopositive for FGF‐1, FGF‐2, and FGFR‐1. In five cases of mucoepidermoid carcinoma (MC), all three types of tumour cells including epidermoid cells, mucous cells, and intermediate cells expressed immunoreactive FGF‐1, FGF‐2, and FGFR‐1. In these malignant salivary gland tumours, increased expression of FGFR‐1 correlated with the intensity of both FGF‐1 and FGF‐2 immunoreactivity. In contrast to malignant salivary gland tumours, eight cases of normal salivary gland showed negative immunostaining for FGF‐1, FGF‐2, and FGFR‐1 while four cases were weakly immunoreactive for FGF and its receptor. These results demonstrate that malignant salivary gland tumours overexpress FGF‐1, FGF‐2, and FGFR‐1 compared with normal salivary glands and suggest that these growth factors may play an important role in facilitating neoplastic progression in human salivary glands.

Malignant tumor cell lines produce interleukin-1-like factor

SummarySixty-four malignant cell lines were examined for interleukin-1 (IL-1) activities in their conditioned medium using thymocyte and fibroblast proliferation assays. Sixteen cell lines showed high IL-1 activity. Comparison of these activities with human IL-1 showed similarity between some biological properties. However there was no correlation between cell origin and IL-1 activity. These results suggest the possibility that most malignant cells may produce an IL-1-like factor.

Growth and differentiation of periodontal ligament-derived cells in serum-free defined culture

SummaryWe have developed a serum-free medium for the growth and differentiation of periodontal ligament-derived cells (PLC). In addition, the expression of both fibroblast growth factor (FGF) and FGF receptor (FGFR) in the PLC was investigated by immunohistochemical examination, heparin affinity chromatography (HAC), and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Optimal growth of the cells was achieved in Iscove’s modified Dulbecco’s medium supplemented with insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, sodium selenite, and oleic acid in type-I collagen-coated dishes. Both FGF-1 and FGF-2 stimulated cell growth and inhibited differentiation as measured by inhibition of alkaline phosphatase activity of the cells. An immunohistochemical analysis of FGF-1 and FGF-2 revealed that immunoreactive FGF-1 and FGF-2 were detected predominantly in the cytoplasm of growing cells. In addition, perinuclear FGF-1 staining and nuclear FGF-2 staining were observed in the same growing cells. In contrast, a faint diffuse staining of FGF-1 and FGF-2 was detected in cytoplasm of the confluent differentiated cells. The 2.15 M NaCl eluate from HAC of the cell extracts exhibited growth-promoting activities for the PLC, and it also stimulated the growth of human umbilical vein-derived endothelial cells and inhibited binding of [125I]-FGF to its receptors, indicating the cells produced FGFs or FGF-like growth factors. RT-PCR analysis revealed that the cells expressed FGFR-1 mRNA but not mRNAs for FGFR-2, FGFR-3 and FGFR-4 mRNA. These results suggest that the FGF-FGFR-1 system plays an important role in the growth and differentiation of periodontal ligament-derived cells.

Expression of fibroblast growth factor-1 (FGF-1), FGF-2 and FGF receptor-1 in a human salivary-gland adenocarcinoma cell line : Evidence of autocrine growth

Fibroblast growth factor‐1 (FGF‐1) and FGF‐2 are heparin‐binding polypeptides which express potent mitogenic properties in neoplastic cells. In the present study, we have examined the contribution of endogenous FGF‐1 and FGF‐2 to the autocrine growth of HSY human salivary‐gland adenocarcinoma cells in vitro. Using specific monoclonal antibodies against FGF‐1 and FGF‐2, immunohistochemical analysis of HSY cells revealed strong expression of both FGF‐1 and FGF‐2 in the cytoplasm and nucleus. Consistent with these data, 2 molecular mass species of FGF‐1 (16 and 18 kDa) and 3 FGF‐2 (18, 24 and 27 kDa) were identified in HSY cells by Western‐blot analysis. Scatchard analysis of FGF binding sites on HSY cells indicated the presence of 23,000 [125I] FGF‐1 binding sites/cells with a dissociation constant (KD) of 178 pM and 13,000 [125I]FGF‐2 binding sites/cell with a KD of 102 pM. In addition, HSY cells were shown to express the mRNA for FGF receptor‐I (FGFR‐1) by reverse transcription‐polymerase chain reaction (RT‐PCR), confirming the existence of high‐affinity FGF binding sites. The influence of endogenous FGF‐1 and FGF‐2 on HSY cell growth was evaluated by suppressing the expression and activity of FGF by using anti‐sense oligonucleotides and neutralizing antibodies. The addition of 50 μM FGF‐1‐specific anti‐sense oligonucleotides to HSY cells resulted in a 61% inhibition of cell growth, while 50 μM FGF‐2‐specific anti‐sense oligonucleotides resulted in a 76% inhibition. These effects were dose‐dependent and specific, since sense oligonucleotides were ineffective in inhibiting HSY cell growth at the same concentration. Furthermore, HSY cell growth was suppressed in the presence of anti‐FGF‐1 or anti‐FGF‐2 neutralizing antibody, resulting in a 58% inhibition at 8 μmg/ml. Our observations suggest that FGF‐1 and FGF‐2 may act as autocrine regulators by interacting with FGF receptors on HSY cells.

An alternative method for the isolation of NS-1 hybridomas using cholesterol auxotrophy of NS-1 mouse myeloma cells

SummaryWe have used the cholesterol auxotrophy of NS-1 mouse myeloma cells as the basis for selecting NS-1 hybridomas. The outgrowth of nascent NS-1 hybridomas in cholesterol-free serum-free medium was 3- to 9-fold more efficient than that in HAT medium and resulted in 3- to 13-times as many antigen-reactive hybridoma wells. This method of hybridoma selection can be applied with any sterol-dependent parent cell line. Hybridomas established under serum-free culture conditions were growth inhibited by fetal calf serum.

Effect of fibroblast growth factor-1 on the three-dimensional growth and morphogenesis of human salivary gland epithelial cells embedded in collagen gels

Dear Editor: The induction of epithelial tubular morphogenesis and glandular histogenesis occurs in vivo during the fetal development of the salivary glands (6). Several in vitro studies have shown that the culture in a three-dimensional collagen gel of epithelial cells from thyroid (9), mammary glands (10), or kidney (8) promotes their organization into tubelike structures. The epithelial structures can mimic the dichotomous branching morphogenesis of tubules and the preliminary steps of glandular development. In fact, the morphogenesis of epithelial tissues can be controlled by the cell-extracellular matrix interactions and by soluble factors released by autocrine and paracrine activation of the epithelial and adjacent mesenchymal cells (3). Fetal salivary gland development is known to correlate with the expression of fibroblast growth factor-1 (FGF-1) (2), which has numerous effects on cellular growth and differentiation in a wide variety of epithelial cells (1). In this report, we cultured human salivary gland epithelial (HSGE) cells within a collagen gel in a serum-free defined medium and found that the sustained growth and duct formation of these cells occurred in the presence of FGF-1. HSGE cells were isolated from labial salivary glands (7). The three-dimensional collagen gels were prepared by mixing 0.4 ml of type I collagen solution (Nitta Gelatin, Tokyo, Japan) and 0.05 ml of 10)< concentrated RD153 medium (RPMI1640:DMEM: MCDB 153; 1:1:2 vol/vol/vol) and 0.05 ml of reconstitution buffer (0.2 M HEPES, 0.08 M NaOH). Collagen (0.5 ml), containing 106 cells, was overlaid on a 0.5 ml base of gelled collagen in each well of 24 well tissue culture plates (Falcon, Mountain View, CA). Cultures were fed with serum-free RD153 medium containing insulin at 10 #g/ml, transferrin at 10 #g/ml, 10/.tM 2-mercaptoethanol, 10 ttM 2-aminoethanol, 10 nM selenite (Sigma Chemical Co., St. Louis, MO) with 20 ng/ml of FGF-1 (Upstate Biotechnology, Lake Placid, NY) and heparin (1000-fold excess by weight). The medium was changed every 3 d. The growth kinetics and morphogenesis was analyzed on Days O, 7, and 14. The number of cells in each well was estimated using a hemocytometer after digestion of collagen with 40 U/ml collagenase (type IA-S, Sigma) followed by a 0.25% trypsin digestion. At the same time, HSGE cells in a collagen gel were fixed with 10% formalin and processed for paraffin embedding, sectioning and stained with hematoxylin and eosin (H-E). In addition, HSGE cells in paraffin sections were immunohistochemically examined using avidin-biotin-peroxidase technique (4) with rabbit polyclonal antisecretory component antibody and antilactoferrin antibody (Dako A/S, Copenhagen, Denmark). As shown in Fig. 1, the growth of HSGE cells in collagen gels was significantly enhanced by FGF-1 in a concentration dependent manner in the presence of heparin. A sixfold increase in cell number could be accomplished at 20 ng/ml of FGF-1 in 14 d. Morphologically, the embedded HSGE cells extended duct-like structures into the collagen matrix overtime. On Day 0, isolated cells and small cell aggregates were seen dispersed within the collagen matrix (Fig. 2 A,B). On Day 7, ductal projections initiated in culture (Fig. 2 C), creating three-dimensional duct-like structures (Fig. 2 D), which became extensive with time (Fig. 2 E,F). When FGF-1 was omitted from medium, HSGE cells did not form duct-like structures; they remained either individual or they formed small clusters (data not shown). Immunohistochemical analysis revealed the presence of secretory component (Fig. 3 A) and lactoferrin (Fig. 3 B) in HSGE cells forming duct-like structures. These polypeptides have been reported to be present in salivary gland duct ceils (5). The results reported here provide evidence that human salivary gland epithelial cells were capable of reconstructing epithelial ductlike structures similar to developing salivary glands when cells were

Monoclonal antibodies against human oral squamous cell carcinoma reacting with keratin proteins

Two mouse monoclonal antibodies (MoAbs), B10 and 1H5, were generated by fusing mouse myeloma NS‐1 cells with spleen cells from a BALB/c mouse immunized with Ueda‐1 cells derived from human squamous cell carcinoma (SCC) of the floor of the mouth. Immunohistochemical analysis revealed that these MoAbs recognize the filamentous components of cytoplasm which were protein in nature. While the pattern of antigen distribution in various cell lines was not cell‐type specific, reactivity of these antibodies with tissue sections was informative. MoAb 1H5 was preferentially reactive with well‐differentiated squamous cell carcinoma, however, reaction with adenocarcinoma was observed infrequently. This antibody also preferentially reacted with the spinous layer of normal stratified squamous epithelium. MoAb B10, however, was reactive with nonepithelial tissues as well as with epithelial ones, and its level of binding bore no relationship to the grade of histologic malignancy. SDS‐PAGE and Western blotting analysis, using cytokeratin extracts of Ueda‐1 cells and human epidermis, demonstrated that MoAb B10 reacted with a wide range of keratin proteins of 65–67K, 58K, 56.5K, 56K, 52K, 50K, 48K, 45K, 40K, 38K, 36K, and 34K molecular weight (MW), while MoAb 1H5 reacted with keratin proteins of 65–67K, 58K, 56.5K, 56K, 52K, 48K, and 34K MW. These results suggest that MoAb 1H5 may recognize keratin subfamilies related to squamous differentiation, whereas MoAb B10 recognizes a wide range of keratin proteins, and may even react with other kinds of intermediate filament proteins (IFPs).

Effects of insulin and transferrin on the generation of lymphokine-activated killer cells in serum-free medium

We have developed a serum-free medium designated RDSF for the generation of LAK cells based on RD6F medium, which was originally developed as a serum-free medium for the growth of myeloma and hybridoma cells. The cytotoxic activity of LAK cells generated in RDSF against Raji, K562 and oral cancer cells, is 3-4 times that of LAK cells generated in medium containing 10% human type AB serum. RDSF medium consisted of nutrient mixture supplemented with transferrin, 2-aminoethanol, 2-mercaptoethanol, sodium selenite and interleukin-2. In this study, we have found that insulin which has been shown to be the most important polypeptide hormone in serum-free media for animal cells, inhibited the generation of cytotoxic activity of LAK cells cultured from peripheral blood lymphocytes. In addition, we found that transferrin was an essential component for the growth and generation of LAK cells in serum-free culture. These results suggest that RDSF may be useful in adoptive immunotherapy for cancer as well as for studying factors involved in the growth and differentiation of LAK cells.

Release of fibroblast growth factor-1 by human squamous cell carcinoma correlates with autocrine cell growth

SummaryA squamous cell carcinoma cell line Nakata proliferated in serum-free culture and was not responsive to exogenous fibroblast growth factor-1 (FGF-1). Immunostaining revealed that Nakata cells expressed FGF-1 in their cytoplasms and nuclei. Two molecular mass species of FGF-1 (16 and 18 kDa) were identified in cell extracts by Western blot. These cells also expressed high-affinity FGF-1 binding sites (Kd=360 pM, 28 000 sites/cell). The results of cross-linking with [125I]FGF-1 demonstrated the presence of two bands with molecular masses of 160 and 140 kDa. The addition of FGF-1 specific antisense oligonucleotides at 25 µM to Nakata cells resulted in an 82% inhibition in cell growth and suppressed FGF-1 expression. This effect was dose-dependent and specific, because sense oligonucleotides were ineffective in inhibiting cell growth. In addition, Nakata cell growth was suppressed by an anti-FGF-1 neutralizing antibody, which resulted in a 52% inhibition at 8 µg/ml. These results demonstrate that Nakata cells produce FGF-1, and indicate that this growth factor acts in an autocrine manner by interacting with FGF-1 binding sites on Nakata cells.

Total upper lip reconstruction using a free radial forearm flap incorporating the brachioradialis muscle: Report of a case

One-stage reconstruction of the upper lip using a free radial forearm flap was successfully performed with excellent functional and cosmetic results. The free radial forearm flap, including the vascularized and innervated brachioradialis muscle, has a very wide potential for reconstruction of the lip defects due to carcinoma.