Daichi Nakata

Enhancement of tumorigenic, metastatic and in vitro invasive capacity of rat mammary tumor cells by transforming growth factor-β

We investigated the effects of transforming growth factor-beta (TGF-beta) on biological behavior of a weakly malignant rat mammary carcinoma ER-1 cell line. TGF-beta enhanced the tumorigenic and metastatic capacity of ER-1 cells and their in vitro invasiveness to rat mesothelial and endothelial cell. Further cell biological analysis indicated that the increased invasive and metastatic capacity of ER-1 cells by TGF-beta was due to the increase in cell motility and adhesion to the mesothelial and endothelial cell monolayers. Thus, it is suggested that TGF-beta acts on ER-1 cells as a progression-enhancing factor which stimulates their adhesive and motile activities.

Adenovirus Type 5 E1A Immortalizes Primary Rat Cells Expressing Wild-Type p53

Adenovirus (Ad) E1A induces apoptosis in cells expressing wild‐type p53, and stable transformation by Ad E1A requires the co‐introduction of an anti‐apoptotic gene such as Ad E1B 19K. Thus, cells immortalized by Ad E1A alone might have lost functional p53. In order to analyze the p53 in rat cells expressing Ad E1A, we established rat cell lines by transfecting primary rat embryo fibroblast (REF) and baby rat kidney (BRK) cells with cloned Ad5 E1A. By using a yeast functional assay, we analyzed p53 in six primary REF and three BRK cell lines immortalized by Ad5 E1A as well as five spontaneously immortalized rat cell lines (REF52, NRK, WFB, Rat‐1 and 3Y1). The yeast functional assay revealed that all of the spontaneously and Ad5 E1A‐immortalized rat cell lines except for 3Y1 expressed wild‐type p53. All of the Ad5 E1A‐immortalized rat cell lines contained p53 detectable by immunoprecipitation. Recombinant adenovirus expressing rat p53 cloned from a REF cell line immortalized by Ad5 E1A, as well as that expressing murine wild‐type p53, induced apoptosis in p53‐null cells in collaboration with E1A. Thus, it is suggested that the mutation of p53 appears to be not frequent in the spontaneous immortalization of primary rat cells, and that the functional loss of wild‐type p53 is not a prerequisite of E1A‐mediated immortalization.

A functional and quantitative mutational analysis of p53 mutations in yeast indicates strand biases and different roles of mutations in DMBA- and BBN-induced tumors in rats

In order to analyze the mutational events and to understand the biological significance of the p53 gene in chemical carcinogenesis, we applied a new yeast‐based p53 functional assay to ovarian tumors induced by 7, 12‐dimethylbenz[a]anthracene (DMBA), as well as to transitional cell carcinomas of the urinary bladder induced by N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine (BBN) in rats. The assay demonstrated that 15 of 19 DMBA induced tumors harbored clonal p53 mutations, which is consistent with the expectations of the “clonal expansion” hypothesis. The majority of the mutations were purine (AG) to pyrimidine (CT) transversions (12/19) on the non‐transcribed (sense) strand (NTS), which is likely to be due to depurination created by DMBA adduct formation on the NTS. In contrast, we found no purine to pyrimidine transversion on the NTS. After cessation of BBN treatment, BBN‐induced multifocal lesions in the bladder contained heterogeneous p53 mutations at an early stage. In the later stage, however, clonal p53 mutations were identified in 4 out of 7 bladders analyzed, conforming with the concept of “field cancerization”. The observed base substitutions were G→A (1/6) or C →T transitions (2/6), and mutations at T (3/6) on the NTS in clonal mutations, together with non‐clonal mutations, showing a preference of C→T to G→A (17 vs. 0). Thus, preferential repair was found in the transcribed strand of the p53 gene, whether modified by DMBA or by BBN carcinogens. Very similar mutation patterns were observed between clonal and non‐clonal mutations in the DMBA‐ and BBN‐induced tumors, indicating that the rat yeast p53 functional assay can be a potential tool for the characterization of in vivo mutation patterns of p53, when modified by chemical carcinogens. Int. J. Cancer 83:700‐705, 1999.

Characterization of the progressive sublines derived from a weakly malignant cloned cell line, ER-1, co-inoculated subcutaneously with a foreign body.

We previously established an experimental model of tumor progression using a weakly malignant rat mammary carcinoma cell line, ER-1. Using this model, we demonstrated that ER-1 cells converted into highly tumorigenic and metastatic cells, ERpP, by s.c. co-inoculation with plastic plates. We here compared in vitro biological properties associated with malignancy of ER-1 cells with those of ERpP cells which were highly malignant when inoculated into syngeneic rats. In vitro growth rate of ERpP cells was higher than that of ER-1 cells under a low nutrient condition. Invasion capacity of ERpP cells to rat lung endothelial cell monolayer or reconstituted basement membrane, Matrigel, was higher than that of ER-1 cells. Migration of ERpP cells toward fibronectin or laminin was also significantly higher than that of ER-1 cells.There was no difference in gelatinolytic or plasminogen activator activity detected in conditioned media between ER-1 and ERpP cells. Furthermore, we found that ER-1 cells communicated better among themselves and with normal fibroblasts through gap junctions compared to ERpP cells. These results suggest that growth advan-tage in a poor nutrient condition, enhancement of cell motility, and loss or decrease of junctional communication may be associated with tumor progression of ER-1 cells.