Makoto Hanazono

Establishment of prostatic cell line "Pro9ad" from a p53-deficient mouse.

We demonstrated that p53‐deficiency is sufficient for immortalization of fetal uterine cells. In the present study, we further extended our previous observations to prostate tissues from a young p53‐deficient adult mouse.

Establishment of an androgen-responsive prostatic cell line “PEA5” from a p53-deficient mouse

We demonstrated that p53‐deficiency is sufficient for the establishment of clonal cell lines from the uterus and prostate. In the present study, we improved cloning methods to establish androgen‐responsive cell lines.

Establishment and characterization of clonal cell lines from the vagina of p53-deficient young mice.

SummaryClonal cell lines have been established from vaginae of prepubertal female p53-/- mice. Because the mouse vagina has a dual origin (the cranial three-fifths derived from the Müllerian duct and the caudal two-fifths derived from the urogenital sinus), both parts were separately subjected to cloning. Sixteen epithelial and two fibroblastic cell lines established from the cranial three-fifths (Müllerian vagina group), and four epithelial and three fibroblastic cell lines were established from the caudal two-fifths (sinus vagina group). They were maintained in Dulbeccos modified Eagle medium and Hams nutrient mixture F-12 containing 10% fetal calf serum and 17β-estradiol at 10−8M. Two cell lines (one epithelial and one fibroblastic) were examined using soft agar assay, but no colonies were formed. The doubling time of cell lines was approaximately 24 h, and all of them divided more than 200 times without crisis, suggesting that they were immortalized. All epithelial cell lines expressed cytokeratin 8. However, the epithelial cell lines expressed cytokeratin 14 and cytokeratin 10 when exposed to medium containing different concentrations of Ca2+. Fibroblastic cell lines expressed vimentin. All epithelial and fibroblastic cell lines expressed estrogen receptor-α protein. This is the first successful establishment of clonal cell lines from the normal mouse vagina, and these lines may provide good models in vitro of the vagina for the study of the mechanism of estrogen action.

Indomethacin Suppresses the Growth of Colon 26, Meth-A and FM3A Tumors in Mice by Reducing the Prostaglandin E2 Content and Telomerase Activity in Tumor Tissues

The antitumor effect of indomethacin on Colon 26, Meth‐A and FM3A tumors was investigated in mice. The prostaglandin E2 content in tumor tissues was assayed to find out if indomethacin acts on tumors, and the telomerase activity in tumors and somatic tissues (testis, liver, spleen and colon) was also monitored during indomethacin treatment. Growth of Colon 26, Meth‐A and FM3A tumors was significantly (P < 0.001‐0.05) suppressed by indomethacin compared to the untreated controls. The prostaglandin E2 content in the three tumors was markedly (P < 0.001) reduced by indomethacin. Telomerase activity in Colon 26 and FM3A tumors was significantly (P < 0.001) lower than that of untreated tumors (80% and 45% decrease versus the controls, respectively), and the activity in Meth‐A tumor was slightly decreased (10% decrease versus the control) by indomethacin. Telomerase activity in the somatic tissues was not significantly affected by indomethacin. In summary, this study shows the effectiveness of indomethacin as an antitumor agent against three types of tumors, and suggests that indomethacin affects telomerase activity in tumors in vivo.

Indomethacin preferentially augments 5-fluorouracil cytotoxicity in Colon 26 tumors by increasing the intracellular inflow of 5-fluorouracil

AbstractBackground. The antitumor effect of indomethacin has been well documented in cancer patients and in animals with transplanted tumors. Indomethacin has also been regarded as a biological response modifier. Therefore, we presumed that indomethacin could be a modulator of antitumor agents, and this study was done to determine whether indomethacin modified the cytotoxicity of selected antitumor agents. Methods. The effect of indomethacin on the antitumor activity of 5-fluorouracil (5FUra), cis-platinum (CDDP), and etoposide in Colon 26 tumor cells was evaluated in vitro by MTT assay. Results. Indomethacin did not show a cytotoxic effect on the tumor cells at pharmacological concentrations. Indomethacin preferentially modulated the cytotoxicity of 5FUra, while it did not affect the cytotoxicity of CDDP and etoposide. Several fold indomethacin (0.01 and 0.1 μg/vol) enhanced 5FUra cytotoxicity in tumor cells. Indomethacin increased the 5FUra content in the tumor cells by 80% over the control through increasing the inflow of 5FUra, rather than by prohibiting the efflux of 5FUra. Conclusion. Indomethacin appears to be a candidate for augmenting 5FUra cytotoxicity in tumor cells by facilitating the intracellular uptake of 5FUra. This study suggests the effectiveness of indomethacin in reducing the dosage of 5FUra or in augmenting 5FUra cytotoxicity when given at the same dose.

Indomethacin acts as an antitumor and anticachexic agent in colon 26-bearing CDF1 mice

BackgroundThis study uses Colon 26-bearing CDF1 mice to assess the pharmacologic actions of indomethacin on tumor growth and cancer cachexia. The effect on tumor growth was evaluated by tumor weight, and the effect on cachexia was screened by changes in body weight and food intake. Additional indices measured included serum levels of immunosuppressive acidic protein and interleukin-6, and prostaglandin E2 content in the tumor tissues.MethodsIndomethacin (1.0 mg/kg body weight, by oral gavage twice per day) was given to 2 groups of mice, group B (long-term therapy) for 21 days, and group C (short-term therapy) for 7 days. A control group (A) received twice-daily oral gavage of the vehicle alone.ResultsMean tumor weight was significantly reduced in groups B and C, compared to the control group. Group B had significantly less body weight loss, and true body weight increased significantly in group C mice from day 17, as compared with the control mice. Serum levels of immunosuppressive acidic protein were significantly reduced in both groups B and C, when compared to the nontreated controls. Serum levels of interleukin-6 were also significantly reduced in both groups B and C, compared to the controls. Prostaglandin E2 content in the tumor tissues was significantly lower in both groups B and C than it was in the controls.ConclusionIndomethacin was shown to act as an antitumor and anticachexic agent. These findings suggest a potential role for indomethacin as a therapeutic agent for cancer.

Dexamethasone Inhibits the Growth of a Uterine Cell Line Derived from p53-Deficient Mice

Abstract UE8 is one of several uterine cell lines established from p53-deficient fetal female mice. UE8 exhibits a typical epithelial morphology in culture; it is strongly positive for cytokeratin, but negative for vimentin. Immunoblot analysis confirmed that UE8 has glucocorticoid receptors. It grows actively in medium supplemented with 3% heat-inactivated, dextran-coated charcoal-treated fetal calf serum. Dexamethazone (DEX) inhibited its growth dose-dependently at concentrations between 10−6and 10−9 M. The inhibition by DEX was further examined in chemically defined medium (CDM): DMEM/F12 containing transferrin (10 μg/ml), selenium (10−8 M) and 0.1% bovine serum albumin. DEX at 10−6 M inhibited the growth of UE8 in CDM or in CDM supplemented with insulin-like growth factor-1 (IGF-1: 10 ng/ml), but there was no inhibition by DEX(10−6M) in the presence of epidermal growth factor (10 ng/ml). Thus, DEX inhibits the growth of a uterine epithelial cell line derived from p53-deficient mice in vitro, and also suppresses IGF-1-induced proliferation.

ICI 182,780 Stimulates the Growth of a Uterine Cell Line Derived from p53-Deficient Mice

Abstract UE8 is a uterine epithelial cell line established from p53-deficient fetal female mice. UE8 exhibits a typical epithelial morphology in culture and is strongly positive for cytokeratin, but negative for vimentin in immunocytochemistry. UE8 shows an active growth in a phenol red free 1:1 mixture of Dulbeccos modified Eagles medium and Hams nutrient mixture F-12 supplemented with 3% heat-inactivated and dextran-coated charcoal-treated fetal calf serum. Both immunocytochemistry and immunoblot analyses confirmed that UE8 was negative for the estrogen receptor. Diethylstilbestrol added to the medium at concentrations between 10−6 and 10−8 M had no significant effect on the proliferative rate, and estradiol-17β at 10−6 M was slightly inhibitory. Unexpectedly, however, the“pure anti-estrogen”ICI 182,780 at 10−6 M significantly enhanced cell proliferation. This is the first observation that the“pure anti-estrogen”ICI 182,780 stimulates the growth of uterine epithelial cells without estrogen receptors.