Tadanori Kameyama

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

Non-transformed, but not ras/myc-transformed, Serum-free Mouse Embryo Cells Recover from Growth Suppression by Azatyrosine

The anti‐proliferative effect of azatyrosine, a newly discovered antibiotic from Streptomyces, was examined in Balb/c‐originated serum‐free mouse embryo (SFME) cells and transformed ras/myc SFME cells which have activated human c‐Ha‐ras genes. Azatyrosine suppressed their growth in a concentration‐dependent manner. Growth suppression in both cells was detectable within 2 days after culture with 250 μg/ml azatyrosine. Non‐transformed SFME cells, however, regained rapid growth after 6 days even in the presence of azatyrosine, whereas ras/myc SFME cells did not recover from the suppression. Despite the growth inhibition of ras/myc SFME cells, expression of human ras in the cells was not inhibited by azatyrosine. Meanwhile, SFME cells have the ability to express glial fibrillary acidic protein (GFAP). This expression is induced by serum‐supplemented medium, though the serum inhibits the growth of SFME cells. Azatyrosine did not induce GFAP in ras/myc SFME cells, but inhibited growth. Furthermore, azatyrosine did not induce GFAP in SFME cells, and had no effect upon the expression of GFAP induced by serum in these cells. These results suggest that azatyrosine inhibited the growth of ras/myc SFME cells through a mechanism independent of those involved in growth inhibition and induction of GFAP expression by serum in SFME cells.

Embryoid bodies cultured in in vivo diffusion chambers show reduced tumorigenicity while retaining expression of F9 antigens

Embryoid bodies of the mouse teratocarcinoma OTT6050 were dissociated into single cells and cultured in diffusion chambers implanted into the peritoneal cavities of mice. The syngeneic host mice, into which the cells of embryoid bodies cultured in the diffusion chambers had been injected, survived much longer than those which received the original cells of embryoid body. But in the case of the F9 cells, obtained in the same culture conditions, only a slight decrease in tumorigenicity was observed. By contrast, the F9 antigenic expression was observed on both F9 and embryoid body cells cultured in diffusion chambers. Judging from the determination of adult-type antigenic expressions, the differentiation of the cells in chamber was negligible. These results suggest that the tumorigenic activity of the embryoid body cells cultured in vivo in a diffusion chamber is almost suppressed, but that they continue in an undifferentiated state.

DIFFUSION CHAMBER CULTURE OF A SINGLE EMBRYOID BODY FROM THE TESTICULAR TERATOMA OF STRAIN 129 MOUSE

When single embryoid bodies of teratocarcinoma OTT 6050 were cultured by the diffusion chamber technique in the peritoneal cavity of a mouse, they lost their characteristic three‐dimensional structure early in the culture period and proliferated logarithmically up to the 60th day of culture with a doubling time of 3.7 days, forming cell layers that adhered to the surface of the membrane filters of the diffusion chamber. They continued further to proliferate at a lower rate up to the 80th day of culture. At the 60th day, many round cells, classified by diameter into about three classes, were observed on the membrane filters. The tumorigenicity of these cells derived from the chamber cultures was much less than that of embryoid bodies injected directly into the abdominal cavity, judging from the number of days the mice survived.

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.

In vivo diffusion chamber culture reduces the tumorigenicity of PCC4-aza1 teratocarcinoma cells

The established cells of strain 129 mouse teratocarcinoma PCC4-aza1 were cultured in diffusion chambers inplanted into the mouse peritoneal cavities. This unique culture reduced the tumorigenic activity of PCC4-aza1 cells without changing their F9 or H-2 antigenicity in a way similar to that described previously for embryoid body cells of OTT6050 teratocarcinoma cultured in diffusion chambers. These observations suggest that a reduction in the tumorigenicity of multipotent teratocarcinoma cells is generally not accompanied by a change in expression of F9 and H-2 antigenicity.