Hayao Nakanishi

One differentiated squamous cell carcinoma (SCC) cell line (RSC3‐E2) and two undifferentiated tumor cell lines (RSC3‐LM and RSC3‐E2R) with different metastatic potential were established from a 4‐nitroquinoline N‐oxide (4NQO)‐induced differentiated SCC in F344 rat tongue. The RSC3‐E2 subline was isolated from a parental cell line (RSC3‐P) by single cell cloning in vitro, whereas the RSC3‐LM subline was isolated from a lung metastatic focus after subcutaneous (s.c.) injection of RSC3‐P cells. The RSC3‐E2R cell line was isolated from a lung metastatic focus following s.c. injection of RSC3‐E2 cells after X‐irradiation in vitro. The RSC3‐E2 cell line is keratinpositive and grows as a keratinizing tumor in nude mice, whereas RSC3‐LM and RSC3‐E2R cells are keratin‐negative, vimentin‐positive and form undifferentiated tumors. When s.c. injected into nude mice, the RSC3‐E2 cell line proved to be non‐metastatic, while the RSC3‐LM cell line was metastatic by both hematogenous and lymphogenous routes, and the RSC3‐E2R cell line was metastatic only hematogenously. In vitro relative growth rates and in vitro invasion activity of these cell lines were in the order RSC3‐LM>RSC3‐E2R>RSC3‐E2. Chromosome analysis revealed two peaks with modal chromosome numbers of 83 and 78 for RSC3‐P cells and single peaks at 83, 78 and 56 for RSC3‐LM, RSC3‐E2 and RSC3‐E2R cell lines, respectively. Common structural abnormalities on chromosome 11 were shared by all cell lines. Mutation analysis of the p53 gene using a yeast functional assay demonstrated RSC3‐LM cell line to have a point mutation at codon 269, whereas RSC3‐E2 and RSC3‐E2R had double mutations at codons 106 and 170 on each allele. These results suggest that the two undifferentiated RSC3‐LM and RSC3‐E2R tumor cell lines with different metastatic potential were generated from differentiated SCC cells via different genetic pathways as a consequence of tumor progression in vivo and in vitro, respectively. These cell lines should provide a useful model for understanding mechanisms of hematogenous and lymphogenous metastasis, as well as tumor progression of oral SCCs.

Detection of free cancer cells in the peritoneal cavity by RT‐PCR using carcinoembryonic antigen (CEA) as a target gene is a more sensitive predictor of peritoneal dissemination than conventional cytology in gastric‐cancer patients. Difficulties with this method are the lack of quantitative assessment of free cancer cells and the length of time before completion. To overcome these problems, we have established a rapid and quantitative detection method using a novel real‐time fluorescence PCR system (LightCycler). Using this device with hybridization probes as fluorophores, we detected CEA mRNA in peritoneal washes during surgery (within 3 hr) without any post‐PCR procedure. This method could reproducibly quantitate 10 to 10⁶ CEA‐expressing colon carcinoma cells per 10⁷ peripheral blood leukocytes, a comparable sensitivity to conventional RT‐PCR with a wide dynamic range. Analysis of peritoneal washes from 109 gastric‐cancer patients with this assay revealed relative values of CEA transcripts that correlated well with the depth of tumor invasion (p < 0.01). Average values of CEA transcript in peritoneal washes in patients with cytology (−)/RT‐PCR(−), cytology (−)/RT‐PCR(+) and cytology (+)/RT‐PCR(+) results were 0.64, 1,525 and 6,715, respectively. Moreover, CEA transcripts in peritoneal washes in patients with synchronous peritoneal metastasis were more than 50‐fold higher than in those without metastasis. These results suggest a positive correlation between CEA mRNA levels in peritoneal washes and prognosis. We conclude that real‐time RT‐PCR with hybridization probes is a sensitive, quantitative, specific and rapid method to detect free cancer cells in peritoneal washes. This clinically relevant system is a powerful technique to evaluate the risk of peritoneal recurrence in patients with gastric cancer. Int. J. Cancer 89:411–417, 2000.

Antimetastatic effects of 5-FU and its derivative, 1-hexylcarbamoyl-5-fluorouracil (HCFU) on human gastric cancer micrometastasis and their mode of action were evaluated, using a spontaneous lung metastasis model (HY-1) in nude mice. Metastases were first detected in the lung from 4 weeks after subcutaneous transplantation, growing intravascularly and forming micrometastases at 100% incidence by 6 weeks after implantation. Lung metastasis in mice bearing subcutaneous tumors was significantly inhibited by HCFU at doses of 100–150 mg kg⁻¹ day⁻¹ without severe toxic side-effects, when orally administered three times per week either from week 4 or week 6 to 9 weeks after implantation. Spontaneous lung metastasis was also inhibited by the administration of 5-FU, but to lesser extent than with HCFU at equimolar low doses. Apoptosis within primary tumors and lung metastatic foci, as detected by the terminal-deoxynucleotidyltransferase-mediated dUTP nick-end labeling method, was found to be significantly enhanced by HCFU as well as 5-FU administration at doses of more than 100 mg kg⁻¹ day⁻¹ and 50 mg kg⁻¹ day⁻¹ respectively. However, proliferating activity of the metastatic foci, as evaluated by MIB-1 immunostaining, was not significantly suppressed by HCFU or 5-FU treatment. Furthermore, polymerase chain reaction analysis using human specific primers for the β-globin gene, which proved to be capable of detecting 10 tumor cells/ml mouse blood, revealed that circulating tumor cells in the peripheral blood of mice bearing primary tumors were reduced by HCFU or 5-FU administration. These results indicate that circulating tumor cells in blood and micrometastases in the lung are sensitive to these chemotherapeutic agents, and suggest that the anti-metastatic effect of these agents is mediated, at least in part, by enhanced apoptosis rather than by inhibition of cell proliferation.