Ming-Yang Yeh

Cloning and characterization of a novel retinoid-inducible gene 1(RIG1) deriving from human gastric cancer cells.

Retinoids exert wide-spectrum anti-tumor activities, which are mediated via the induction of growth arrest, differentiation or apoptosis. To determine whether the effects of retinoids are mediated by specific gene activation or repression, SC-M1 CL23 gastric cancer cells, pretreated with either vehicle alone or all-trans retinoic acid (10 microM) for 1 day, were analyzed using the technique of differential display. A novel retinoid-inducible gene 1 (RIG1) was isolated. The full-length RIG1 cDNA contained 768 base pairs and encoded a protein of 164 amino acids with a molecular weight of 18 kDa. The RIG1 gene was ubiquitously expressed in normal tissue, and its expression was positively associated with cellular density. Nucleotide sequence analysis demonstrated that the RIG1 gene was similar to a recently-isolated TIG3 gene, and displayed 54% nucleotide sequence homology with a type II tumor suppressor gene H-REV-107-1. RIG1 cDNA, however, contained an extra 32 base pairs located at its 5 end and revealed three base pair differences for the remaining sequences leading to two amino acids substitution between the two encoded proteins. All-trans retinoic acid increased the level of RIG1 mRNA in a time- and concentration-dependent manner in SC-M1 CL23 gastric cancer cells. This was not observed for the H-REV-107-1 gene. The RIG1 regulation was related to cellular retinoid sensitivity. Both retinoic acid receptor alpha- and retinoic acid receptor gamma-selective agonists increased RIG1 mRNA level, and the retinoid x receptor-selective agonist potentiated this regulation. In conclusion, the cDNA of a novel retinoid-inducible gene RIG1 has been cloned. This gene is regulated by retinoic acid through the heterodimer of retinoic acid receptor and retinoid x receptor.

Expression of nuclear retinoid receptors in normal, premalignant and malignant gastric tissues determined by in situ hybridization

Retinoids exhibit multiple functions through interaction with nuclear retinoid receptors and have growth-suppressive activity on gastric cancer cells. To better understand the roles of nuclear retinoid receptors during gastric carcinogenesis, we have used in situ hybridization to investigate expression of retinoic acid receptors (RARs) and retinoid x receptors (RXRs) in premalignant and malignant formalin-fixed paraffin-embedded gastric tissues. Histological sections of eight normal, 17 distal normal and nine gastric cancer tissues were hybridized with non-radioactive RNA probes for subtypes of RAR and RXR. Expression of RARα, RARβ, RARγ, RXRα and RXRβ was found in most cell types in gastric mucosa tissues from normal individuals as well as in distal normal tissues from cancer patients. Expression of RARα and RARβ were found in three and seven cancer tissues, respectively, and levels of RXRα mRNA were significantly decreased in poorly differentiated cancer tissues. Among the five investigated nuclear retinoid receptors, only expression of RARα mRNA was significantly decreased in intestinal metaplasia, dysplasia and cancer tissues when compared to adjacent normal tissues. In conclusion, normal gastric mucosa expressed both RARs and RXRs, which supports the physiological role of retinoic acid on normal gastric mucosa. The decrease in RARα expression in premalignant and malignant gastric tissues suggests a significant role of RARα during gastric carcinogenesis.

In vitro and in vivo Growth Inhibition of SC-M1 Gastric Cancer Cells by Retinoic Acid

Retinoids are differentiating agents that have been used successfully for the treatment of acute promyelocytic leukemia. When combined with interferons, they are active in preventing second malignancies in patients with head and neck cancer. Our previous studies have demonstrated cytostatic effects of alltrans-retinoic acid (tRA) on SC-M1 gastric cancer cells in vitro. The activity of tRA and 13-cis-retinoic acid (cRA) on SC-M1 cells was compared both in vitro and in vivo in this study. Measurement of total cellular DNA was used to determine cell growth in vitro. The effect of retinoic acid on tumor growth was evaluated by implanting sustained release tRA or cRA pellets into athymic nude mice. The results showed that tRA was more potent than cRA in suppressing the growth of SC-M1 gastric cancer cells in vitro. Both tRA and cRA were effective in suppressing the growth of SC-M1 tumors in athymic nude mice. No change in the differentiation status and cell cycle phase distribution in excised tumors was observed. Side effects such as bone fractures and weight loss were observed in mice of both treatment groups. The results suggest that retinoic acid may provide therapeutic advantages for the treatment of gastric cancer.

All-trans retinoic acid decreases susceptibility of a gastric cancer cell line to lymphokine-activated killer cytotoxicity.

All-trans retinoic acid (RA) was previously shown to regulate the growth of gastric cancer cells derived from the cell line SC-M1. This study was designed to investigate the effect of RA on the sensitivity of SC-M1 cells to lymphokine-activated killer (LAK) activity. RA at the concentration range of 0.001-10 microM was shown to induce SC-M1 cells to exhibit resistance to LAK activity in a dose-dependent manner. A kinetics study indicated that a significantly increased resistance was detected after 2 days of co-culturing SC-M1 cells with RA and reached a maximum after 6 days of culture. Similar results were obtained from two other cancer cell lines: promyelocytic leukaemia HL-60 and hepatic cancer Hep 3B. A binding assay demonstrated that the binding efficacy between target SC-M1 cells and effector LAK cells was not altered by RA. Flow cytometric analyses revealed that RA exhibited no effect on the expression of cell surface molecules, including HLA class I and class II antigens, intercellular adhesion molecule-1 and -2, and lymphocyte function antigen-3. Cell cycle analysis revealed that culture of SC-M1 cells with RA resulted in an increase in G0/G1 phase and a decrease in S phase, accompanied by a decrease in cyclin A and cyclin B1 mRNA as determined by Northern blot analysis. Additionally, RA was shown to enhance the expression of retinoic acid receptor alpha (RAR alpha) in SC-M1 cells, and to have no effect on the expression of RARbeta or RARgamma. Taken together, these results indicate that RA can significantly increase gastric cancer cells SC-M1 to resist LAK cytotoxicity by means of a cytostatic effect through a mechanism relating to cell cycle regulation. The prevailing ideas, such as a decrease in effector to target cell binding, a reduced MHC class I antigen expression or an altered RARbeta expression, are not involved.

Antitumor Activity of Doxorubicin-Monoclonal Antibody Conjugate on Human Bladder Cancer

Doxorubicin (adriamycin) was conjugated via the dextran bridge method to a murine IgG3 monoclonal antibody, 1G3.10, directed against human bladder cancer. The drug-antibody conjugate, prepared from using 25% oxidized dextran as the linker, retained essentially the original immunological activity of the antibody using ELISA as tested against an antigen-positive target cell line (TSGH-8301), which has been shown to express an antigen recognized by the monoclonal antibody 1G3.10. Antitumor effect of the conjugate in vitro was evaluated by its inhibition on 3H-uridine incorporation into the established human bladder cancer cells. The conjugate exhibited a significantly higher cytotoxicity on target TSGH-8301 cells than that by a control antibody-doxorubicin conjugate prepared identically from an irrelevant mouse IgG3 monoclonal antibody. No apparently different cytotoxicity was detected on control antigen-negative bladder tumor cells of J82 between these two drug-antibody conjugates. Verapamil, a calcium channel blocker, enhanced the in vitro cytotoxicity of doxorubicin-1G3.10 monoclonal antibody conjugate. Results obtained from in vivo evaluation using xenografted target TSGH-8301 bladder tumor indicated that the 1G3.10 monoclonal antibody conjugate containing doxorubicin injected 4X, i.p., significantly inhibited TSGH-8301 bladder tumor growth in nude mice, whereas free monoclonal antibody, free drug and the mixture of both showed only moderate inhibition of tumor growth as compared to the untreated control. Verapamil also enhanced in vivo antitumor activity of the conjugate. There was no side effect (weight loss) detected on the conjugate-treated mice. Results obtained from in vivo evaluation using xenografted control J82 bladder tumor showed no specific antitumor activity as exhibited by doxorubicin-1G3.10 monoclonal antibody conjugate in comparison with free drug, mixture of drug and antibody without conjugation, or doxorubicin conjugated to the irrelevant antibody. These results suggested that doxorubicin conjugated with bladder tumor associated monoclonal antibody could be useful as a potentially cytotoxic agent in immunochemotherapy of human bladder cancer.

In Vitro Radiosensitivity Assay of Human Urologic Malignancies

The in vitro radiation sensitivity of cells isolated directly from human urologic tumor surgical specimens was studied using the liquid medium culture cultre and trypan blue dye exclusion test. Aerobic cell survival surves were achieved for thirty-one transitional cell carcinomas, fourteen renal cell carcinomas, five testicular tumors, two prostate carcinomas, one squamous cell carcinoma of the kidney and one retroperitoneal fibrohistiocytoma. The Do (0.10-3.20 Gy), Dq(0.10-1.90 Gy), n (1.0-7.0) as well as the surviving fraction at 2.0 Gy (0.001-09) differed significantly among individual tumors of the same histological type. Neither of these parameters was significantly different for the transitional cell carcinoma, renal cell carcinoma, testis and prostate tumor categories. However, the only case of squamous cell carcinoma of the kidney presented the highest surviving fraction at 2 Gy (0.9) than the other tumors. Three out of four primary testicular tumors (three seminomas, one yolk sac tumor) showed surviving fractions at 2 Gy lower than 0.20. (J Uorl R.O.C., 1: 108-217,1990)

Transplantation of Human Transitional Cell Carcinoma in the Nude Mouse Bladder: Comparison between Electrically and Chemically Injured Urothelium

Human transitional cell carcinoma was successfully transplanted and established into the bladder of nude mouse via techniques of electrically or chemically (N-nitro-N-methyl urea) injured urothelium with instillation of tumor cells from a human bladder cancer cell line TSGH-8301. The mice with chemically injured urothelium had higher mortality (60% vs 20%) and morbidity rate than mice with electrically treated urothelium. Tumor taking rate was much higher in the lelctrically treated mice (63%) than chemically treated mice (25%) and control group (10%). This model makes animal studies for human bladder cancer possible and more physiological.