Moriaki Kusakabe

Tenascin-C regulates angiogenesis in tumor through the regulation of vascular endothelial growth factor expression

In order to verify whether tenascin‐C (TN‐C) is involved in angiogenesis as an extracellular signal molecule during tumorigenesis, cancerous cell transplantation experiments and coculture experiments were carried out, focusing on the regulation of vascular endothelial growth factor (VEGF). The A375 human melanoma cells introduced the GFP gene (A375‐GFP), implanted subcutaneously into BALB/cA nude (WT) and TN‐C knockout BALB/cA nude (TNKO) congenic mice. Furthermore, coculture experiments between A375‐GFP and embryonic mesenchyme, which was prepared from both genotypes, were carried out to investigate the molecular mechanism in the cell‐cell interactions. Both the content of TN‐C and that of VEGF in the tumor and the conditioned medium were analyzed by the sandwich ELISA method. Seven days after transplantation of the A375‐GFP, capillary nets became far more abundant in the tumors grown in WT mice than those in TNKO mice. Interestingly, VEGF and TN‐C expressions showed antithetical expression patterns between the tumors in WT mice and those in TNKO mice. This peculiar phenomenon seems to be caused by a time lag prior to the onset of the mesenchymal regulation for the TN‐C expression of A375‐GFP. The coculture experiments revealed that WT mesenchyme had a much stronger effect than TNKO mesenchyme on both TN‐C and VEGF expression. However, the defects of TNKO mesenchyme were restored in all cases by additional TN‐C. These results clearly indicated that the expressions of both TN‐C and VEGF depend on the surrounding mesenchyme, and that the function of mesenchyme is regulated by its own mesenchymal TN‐C. In conclusion, the present data suggest that the matrix microenvironment organized by the host mesenchyme is very important for angiogenesis in tumor development.

Clonality of Urogenital Organs as Determined by Analysis of Chimeric Mice

Though the first mammalian chimera was reported in 1961, suitable markers for different animal strains which are easily detectable in histological sections of all or most organs have not existed. Chimeric mice were produced having an excellent histological marker, the C3H antigen, which is strain-specific and fulfills all the criteria for an ideal strain-specific histological marker. Using male and female C3H-Balb/c chimeric mice we examined epithelial cells of urogenital organs and their morphological or functional units, such as the glomerulus, to determine whether individual organs and their morphological subunits were monoclonal or polyclonal in origin. We found that the epithelial parenchyma of most male and female urogenital organs (the prostate, seminal vesicle, epididymis, ovaries, vagina, kidney, ureter and bladder) and their morphological subdivisions were derived from cells of both input strains, indicating a polyclonal origin for each organ and/or organ component. A notable exception was the uterus in which all individual uterine glands examined (n = 403) were found to be either entirely Balb/c or entirely C3H, indicating a monoclonal origin. The clonality of urogenital structures is discussed in terms of the morphogenesis of the urogenital system.

Invasion of Melanoma in Double Knockout Mice Lacking Tenascin‐X and Tenascin‐C

The roles of extracellular matrix glycoproteins belonging to the tenascin family in the regulation of tumor cell proliferation, invasion, and metastasis are not known. To address this issue, we generated tenascin‐X (TNX) and tenascin‐C (TNC) double knockout mice and compared findings in these mice with those in single knockout (TNX+/+TNC‐/‐ and TNX‐/‐TNC+/+) mice. We investigated the proliferation and invasion of B16‐BL6 melanoma cells after these cells had been injected into the footpads of mice in each group. The primary tumor size and invasion to the ankle adjacent to the primary tumor site were examined at 35 days after injection of the melanoma cells. The primary tumor size in TNX‐/‐TNC+/+ mice was significantly larger than that in wild‐type mice, but those of TNX+/+TNC‐/‐ and double knockout mice were comparable to that in the wild‐type mice. On the other hand, invasion to the ankle was obviously promoted in TNX‐I‐ TNC+/+ and double knockout mice compared with that in the wild‐type mice, but invasion to the ankle in TNX+/+TNC‐/‐ mice was only slightly promoted. Gelatin zymography confirmed increased matrix metalloproteinase (MMP)‐9 activity in the dorsal skin of TNX‐/‐TNC+/+, TNX+/+TNC‐/‐ and double knockout mice. However, the amounts of MMP‐9 mRNA in the dorsal skins of all mice were almost the same, indicating that the increased activity of MMP‐9 in the single and double knockout mice is regulated at the MMP‐9 processing level. These results indicate that MMP‐9 is activated in all TN‐deficient mice, but that TNX exerts a greater effect on tumor invasion than does TNC.