Haruhiko Tokuyama

Bovine colostric transforming growth factor-β-like peptide that induces growth inhibition and changes in morphology of human osteogenic sarcoma cells(MG-63)

TGF-beta like peptide, termed TGF(BC-1), was partially purified from defatted and decaseinated bovine colostrum by a sequence of DEAE-Sephacel chromatography and Sephadex G-50 gel filtration in 1M acetic acid. TGF(BC-1) was distinct from well-known 25K TGF-beta in chemical properties: TGF(BC-1) was sensitive to acid ethanol extraction (Roberts et al., 1980). Its apparent molecular weight ranged from 21k to 11k by gel filtration and it was composed of low MW peptides (15k, 13k, 10k and 7.3k but not 25k) as examined by SDS-PAGE under non-reducing conditions. However, TGF(BC-1) shares some biological properties with the prototype TGF-b. TGF(BC-1) remarkably suppressed growth of osteogenic sarcoma cells (MG-63), and this was intriguingly accompanied by a striking change in morphology.

Expression of placental alkaline phosphatase in gastric and colorectal cancers: An immunohistochemical study using the prepared monoclonal antibody

The authors developed monoclonal antibodies (MoAb) against human placental alkaline phosphatase (PLAP). Four specific MoAb reacting only with PLAP and two nonspecific MoAb reacting equally with isozymes of alkaline phosphatase (hepatic, intestinal, and placental) were obtained. Immunohistochemical staining with the specific MoAb showed that the cell membrane and cytoplasm of cancer cells were stained in gastric and colorectal carcinoma. the incidence of PLAP positivity was 23% (25 of 107) of all gastric carcinomas. Among gastric carcinomas, the 42% (13 of 31) positivity of highly differentiated carcinoma (papillary adenocarcinoma and well‐differentiated tubular adenocarcinoma) was a significantly higher rate than that found in poorly differentiated carcinoma (poorly differentiated adenocarcinoma and signet‐ring cell carcinoma, five of 41, 12%). the incidence of PLAP positivity was 11% (four of 35) in colorectal carcinoma. in contrast, gastric adenoma, intestinal metaplasia, and noncancerous tissue adjacent to cancer did not show staining. These results indicated that expression of PLAP was apt to occur in more highly differentiated gastric carcinoma and was highly specific for carcinoma in the gastrointestinal tract, although its incidence was not high.

Purification and identification of TGF-β2-related growth factor from bovine colostrum

Bovine colostrum contains transforming growth factor (TGF)-beta-like activity. High levels of this activity are found in early colostrum (within 12 h after parturition); however, it decreases rapidly after 30 h. In this study, the activity in early colostrum was purified by a sequence of decaseination, DEAE-Sephacel chromatography, Sephadex G-100 gel filtration, precipitation at neutral pH, further gel filtration and reversed-phase FPLC. A homodimeric 23 kDa protein was identified that retained the TGF-beta-like activity. Its activity was completely neutralized by a specific anti-TGF-beta 2 antibody. These results indicate that the TGF-beta-like growth factor found in bovine colostrum is chemically and antigenically related to TGF-beta 2. The physiological roles of the TGF-beta 2-related growth factor are discussed.

Establishment of a metastatic murine cell line carrying the human c-Ha-ras

Dear Editor: Development of new modalities to control metastasis is an urgent requirement of cancer therapy. However, the available methods have an inherent limitation in detecting and quantifying micro-metastases. It is possible to experimentally detect metastasis if genes of a species are detected in the tissues of another animal. Therefore, a new system needs to be established which consists of: 1) ceils that grow and metastasize in immunocompetent syngeneic animals, a n d 2) cells that have genes which are distinguishable from those of the animal tissues. The combination of r/mHM-SFME-1 cells and Balb/c mice provides a good model system for this application. The r/mHM-SFME1 cells are derived from ras/myc SFME cells transformed by activated human c-Ha-ras and mouse c-myc genes (5,7). Since original serum-free mouse embryo (SFME) cells were established from a Balb/c mouse embryo (3), immunocompetent Balb/c mice are syngeneic for both ras/myc SFME and r/mHM-SFME-1 cells. Here we describe the establishment of the r/mHM-SFME-1 cell line in vitro. One million of G418-resistant ras/myc SFME cells, which were transfected with pSV2-neo by calcium-phosphate co-precipitation (7), were injected subcutaneously into the backs of Balb/c mice. All of mice, which developed solid tumors within 2 months, were sacrificed to check metastases. One of them had metastases in the subaxillary and submaxillary lymph nodes, the lung and the liver. The metastases were excised from each organ and transplanted subcutaneously again into the mice. Only mice that received the tung metastases developed solid tumors and pulmonary metastases. Thereafter, these pulmonary metastases were serially transplanted subcutaneously into Balb/c mice. The solid tumors of the 7th passage were excised under sterile conditions and the cells were then cultured in serum-free medium (3) followed by colonization in soft agar. One of the clones derived from a colony was designated as r/mHM-SFME1. The r/mHM-SFME-1 cells were no longer resistant to G418. The profiles of the two cell lines in culture are shown in Figure 1. The r/mHM-SFME-1 cells tended to aggregate in culture whereas the ras/myc SFME cells did not. Aggregates always appeared 3 4 days after plating whether in serum-free or in serum-supplemented media and did not disappear upon adding fresh media. The aggregates sometimes detached from cells on dishes so that freely floating cells were observable in aged cultures even maintained by frequent media change. In order to confirm that the r/mI-LM-SFME-1 cells were derived from the ras/myc SFME cells, we determined whether the r/mHMSFME-1 cells had human ras genes. Hind III-digested fragments of DNA from r/mHM-SFME-1 and ras/myc SFME ceils were hybridized with the human c-Ha-ras exon-2 (6). A plasmid pUCC-H-ras, which contains normal human ras gene from placenta, for probe was obtained from the Japanese Cancer Research Resources Bank (JCRB). Six bands were detected in each of the fragments and no significant differences in the band profiles of either fragment were observed (Fig. 2). A faint band detectable in the DNA fragments from non-transformed SFME cells may be due to endogenous

Isolation and characterization of pokeweed mitogen-like phytomitogens from shoriku, Phytolacca esculenta

From saline extracts of Phytolacca esculenta (shoriku) roots, two phytomitogenes were isolated by salting out with (NH4),SO4 and chromatography on DEAE-cellulose and Sephadex G-100 columns. Both fractions were homogeneous on disc electrophoresis and on immunoelectrophoresis. One of these (Fraction E-2) was shown to be similar to pokeweed mitogen in respect to mol. wt (32,000) and amino acid composition. The other (Fraction E-3) was a protein of 18,000 mol. wt. Both fractions had similar biological activities to pokeweed mitogen in their ability to stimulate pig blood lymphocytes in vitro to incorporate tritiated thymidine, and to induce blastoid transformation. Both fractions contained an unusually large amount of cystine, i.e., 18 half-cystine residues % for Fraction E-2 and 22 residues % for Fraction E-3. Although these mitogens were resistant to deproteinizing procedures such as perchloric acid treatment and Sevags procedure, the DNA synthesis-stimulating activity was inactivated by digestion with Pronase E and Nagarse, but resistant to trypsin, chymotrypsin, deoxyribonuclease, ribonuclease, lysozyme and neuraminidase. The activity was stable at acidic and neutral pH (4-7) but unstable at alkaline pH. The activity at pH 7.3 was stabilized by the addition of Ca2+ or Mg2+. On the addition of more than 2 mM of Ca2+, precipitation of mitogen occurred. From the above results the molecular basis of the mitogenic activity of shoriku mitogen is discussed.

Retinoic acid and steroid hormones regulate IgA production by LPS-stimulated murine spleen cells

We examined the regulatory effects of steroid hormones (beta-estradiol and glucocorticoids) on in vitro IgA production. Addition of retinoic acid (RA, 100-500 nM) to the LPS-stimulated spleen cell culture enhanced IgA production (8-22-fold). Simultaneous addition of beta-estradiol, but not testosterone, enhanced the effect of RA synergistically (a further 2-4-fold). In contrast, glucocorticoids inhibited the reaction. The concentration inhibiting IgA production by 50% was 1 nM, 6 nM and 10 nM for dexamethasone, prednisolone and hydrocortisone, respectively. None of the hormones tested alone affected IgA production by LPS-stimulated spleen cells. Hydrocortisone enhanced the IgG1 production by LPS-stimulated spleen cells. This effect was completely abolished by simultaneous addition of RA. These findings indicate that RA can direct the class-switching to IgA in LPS-stimulated spleen cells, and that beta-estradiol and glucocorticoids have positive and negative regulatory effects, respectively, on the IgA production.

Characterization of Newly Established Cells Which Provide an Animal Model for Spontaneous Metastasis

A novel cell line, r/mHM-SFME-1 (r/mHM-1) was established from ras/myc SFME cells transformed by human c-Ha-ras and mouse c-myc genes (SFME cells have been established from a Balb/c mouse embryo in a serum-free culture condition). This cell line was derived from a pulmonary metastasis developed in a Balb/c mouse which had been transplanted subcutaneously with pSV2-neo introduced ras/myc SFME cells. The r/mHM-1 cells had an ability to spontaneously metastasize into the lungs of syngeneic mice when injected subcutaneously, and survival of the mice which received the r/mHM-1 cells was significantly shorter than ones with ras/myc SFME cells. The r/mHM-1 cells grew slowly in vitro than their parental ras/myc SFME ones did, and produced dispersed colonies in agar whereas their parental ones produced packed ones. A urokinase type plasminogen activator activity was detected in the culture fluid in which the r/mHM-1 cells were cultured for 2 days, whereas the activity was not detected in those from the parent ones.