Hiroyuki Sadano

Effect of (−)-epigallocatechin gallate, the main constituent of green tea, on lung metastasis with mouse B16 melanoma cell lines

(-)-Epigallocatechin gallate (EGCG), the main polyphenolic constituent of green tea, inhibits tumor promotion and chemical carcinogenesis in animal experimental systems. Here we report that the peroral administration of EGCG inhibited metastasis of B16 melanoma cell lines, such as B16-F10 and BL6, in both experimental and spontaneous systems.

The Newly Identified Human Nuclear Protein NXP-2 Possesses Three Distinct Domains, the Nuclear Matrix-binding, RNA-binding, and Coiled-coil Domains

Using a monoclonal antibody that recognizes a nuclear matrix protein, we selected a cDNA clone from a λgt11 human placenta cDNA library. This cDNA encoded a 939-amino acid protein designated nuclear matrix protein NXP-2. Northern blot analysis indicated that NXP-2 was expressed in various tissues at different levels. Forcibly expressed green fluorescent protein-tagged NXP-2 as well as endogenous NXP-2 was localized in the nucleus and distributed to the nuclear matrix. NXP-2 was released from the nuclear matrix when RNase A was included in the buffer for nuclear matrix preparation. Mapping of functional domains was carried out using green fluorescent protein-tagged truncated mutants of NXP-2. The region of amino acids 326–353 was responsible for nuclear matrix binding and contained a cluster of hydrophobic amino acids that was similar to the nuclear matrix targeting signal of acute myeloleukemia protein. The central region (amino acids 500–591) was demonstrated to be required for RNA binding by Northwestern analysis, although NXP-2 lacked a known RNA binding motif. The region of amino acid residues 682–876 was predicted to have a coiled-coil structure. The RNA-binding, nuclear matrix-binding, and coiled-coil domains are structurally separated, suggesting that NXP-2 plays important roles in diverse nuclear functions, including RNA metabolism and maintenance of nuclear architecture.

Newly identified type of β actin reduces invasiveness of mouse B16-melanoma

Low metastatic parent B16 melanoma and isolated B16‐F1 cell lines have a third actin designated as βm(Ax:previously). ßm actin is scantily or not at all detected in highly metastatic cell lines, such as B16‐F10 and BL6. To directly assess the physiological role of ßm in phenotypic changes of B16 melanoma, we transfected expression plasmids of βm into B16‐F10 cells. The actin expressed in the transfectants is located largely in cytoskeletal fractios. The transfectants exhibited a larger number of stress fibers and a lower invasiveness than did the recipient cells. Thus, βm actin plays an important role in the organization of actin stress fibers, the result being a decrease in invasiveness of B16 melanoma.

Differential expression of vinculin between weakly and highly metastatic B16-melanoma cell lines

We previously reported on the altered expression of a third actin in mouse‐B16 melanoma associated with malignant progression. White further investigating the relationship of cytoskeletal proteins to malignancy, we found that the expression of vinculin was higher in weakly metastatic B16‐F1 cells than in highly metastatic B16‐F10 cells. By Northern blot analysis, the mRNA expression of vinculin in B16‐F1 was also shown to be higher than in B16‐F10. Immunofluorescence staining showed a clear dotted distribution of vinculin in B16‐F1, but only a weak and diffuse distribution in B16‐F10. The dotted distribution tended to be larger in B16‐F1 and when cultured on Matrigel and fibronectin than on laminin and type IV collagen. An alteration in the expression of vinculin was also observed in other cell systems. Vinculin was detected in both normal 3Y1 and in relatively weakly malignant transformed 3Y1 cell lines, while vinculin was either scarcely detected or not detected at all in more malignant cell lines. These results suggest that the suppression of vinculin is closely related to malignant progression in both the B16‐melanoma and 3Y1 cell systems.

Altered expression of a third actin accompanying malignant progression in mouse B16 melanoma cells

The expression of actin was examined and compared in several mouse B16 melanoma cell lines with different metastatic ability, by the use of two‐dimensional gel electrophoresis or horizontal isoelectric focusing. In the mouse B16 melanoma cell lines, the expression of newly found Ax actin (Mr = 43,000, pl = 5.2) decreased with the increase in in vitro and in vivo selection cycles (F number) for high‐metastatic cells. On the contrary, the metastatic ability of each mouse cell line, assessed by lung colony‐forming ability following iv administration, increased with increase in the F number. The half life of Ax actin was much the same as that of β‐and γ‐actin and the different expressions of Ax actin between the low‐ (F=l) and high‐metastatic (F=10) cell lines were attributed to differences in the rate of synthesis but not in the decay rate of Ax actin. The Ax actin was incorporated into the cytoskeletal fraction with the same efficiency as β‐and γ‐actin. The invasiveness of the cells, assessed in vitro using matrigel, was increased with the decrease in Ax expression. The actin stress fibers, observed staining with rhodamine‐conjugated phalloidin, were organized better in a low‐metastatic cell line (F=l) than in a high‐metastatic one (F = 10). These results suggest to us that depression of Ax actin is involved in disorganization of the cytoskeletal system, the cellular flexibility and motility are enhanced and there is a consequent increase in the invasiveness and metastatic potential.

Reversible transfer of heme between different molecular species of microsome-bound cytochrome P-450 in rat liver

Incorporation of newly synthesized heme into microsome-bound cytochrome P-450 in rat liver was not affected by cycloheximide administration to the animals, indicating that the heme incorporation into cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When the heme of microsomal cytochrome P-450 had been labeled in vivo with delta-[14C]aminolevulinic acid, and then the animals were treated with phenobarbital (PB) or 3-methylcholanthrene (MC), PB-induced or MC-induced form of cytochrome P-450 was found to contain labeled heme derived from preexistent cytochrome P-450. These observations indicated that the heme of microsome-bound cytochrome P-450 is not tightly associated with the protein portion, and exchanges reversibly between different molecular species of cytochrome P-450 in vivo.

Differential expression of smooth muscle α-like actin between benign and malignant human pigment tissues

When examining proteins in human pigment tissues, we found that a third actin-like protein, in addition to beta- and gamma-actin was more frequent and in a larger amount in benign tissues such as blue nevus and nevus pigmentosus, than in malignant melanoma. This third actin-like protein was immunologically stained with monoclonal antibodies reacting with several actin species and specific for smooth muscle alpha-actin. We propose that this third actin-like protein is probably smooth muscle alpha-actin and that different expressions of this third actin may possibly serve as a sensitive biochemical marker for the diagnosis of human malignant melanoma.

Malignant progression of a transformed rat cell line by transfer of the v-fos oncogene.

Transfer of the v-fos oncogene into a rat cell line transformed by Rous sarcoma virus increased both the spontaneous and experimental lung-metastasis. Metastatic ability of each v-fos transferred cell line was dependent on both the manner of integration and transcriptional amount of the v-fos oncogene, but did not correlate with the growth rate in vivo. Expression of the src, myc or ras genes were not altered by transfer of the v-fos gene, except that the myc expression was enhanced in the cell line, which acquired augmentation of growth rate in vivo but not metastatic potential to the lung. Cells of the metastatic lung nodules of each cell line also possessed exogenous fos DNA and the transcripts. These results suggest that v-fos oncogene functions in the transfected cells and causes malignant progression.

Intracellular Localization and Biochemical Function of Variant β-Actin, which Inhibits Metastasis of B16 Melanoma

We analyzed the biochemical nature of βm‐actin protein found in mouse B16 melanoma. When we carried out immunostaining with the antibody specific to βm‐actin, filamentous immunofluorescence was observed in B16‐F1, a low‐metastatic cell line expressing βm‐actin, but not in highly metastatic B16‐F10 that did not express βm‐actin. When a purified actin fraction containing βm‐actin was polymerized and immunoprecipitated with anti‐βm‐actin antibody, the immunoprecipitate contained βm‐, β‐ and γ‐actin. This indicated that the βm‐actin was incorporated into an actin filament together with β‐ and γ‐actin in vitro, and this phenomenon was consistently suggested by cellular double immunostaining with anti‐βm‐actin and common anti‐actin antibody. When the actin fraction containing βm‐actin under a regular depolymerizing condition was subjected to immuno‐adsorption assay using anti‐βm antibody and protein‐A Sepharose, the immunoadsorbed aggregates contained βm‐, β‐and γ‐actin. This indicates that the actin fraction was not completely depolymerized and contained βm‐actin‐containing oligomers, which were too small to be precipitated with anti‐βm‐actin antibody alone. The incomplete depolymerization of the βm‐actin‐containing fraction was also suggested by the much lower DNase 1 inhibition activity of the βm‐actin‐containing fraction than that of β‐ and γ‐actin fraction. Furthermore, a DNase 1 binding assay showed that cytoplasmic supernatant prepared from B16‐F1 under a low‐ionic condition contained less monomeric actin than the cytoplasmic preparation from B16‐F10. These results suggested that βm‐actin protein in B16 melanoma probably inhibits the dynamic conversion between the monomeric and polymerized forms of actin, leading to a decrease in cell motility and consequently the suppression of invasiveness and metastasis.