Takejiro Kuzumaki

Effect of flavonoids on cell cycle progression in prostate cancer cells.

The effect of some flavonoids, which are components of fruits, vegetables, and peas, on the cell cycle progression of human LNCaP prostate cancer cells has been investigated in this study. Genistein arrested the cell cycle at the G2/M phases, which is attributed to the suppression of cyclin B expression. In addition, genistein induced the cyclin-dependent kinase inhibitor p21, which does not depend on p53 activation. Apigenin and luteolin also increased p21 levels, but quercetin did not. Apigenin induced p21 production through a p53-dependent pathway, but luteolin did so in a p53-independent manner. These results suggest that flavonoids are potent regulators of cyclin B and p21 for cell cycle progression, which may play some roles in prevention of carcinogenesis.

Interleukin-6 down-regulates expressions of the aldolase B and albumin genes through a pathway involving the activation of tyrosine kinase

Interleukin-6 plays a key role in mediating acute-phase protein synthesis in hepatocytes. However, the mechanism of how interleukin-6 regulates aldolase B and albumin syntheses in hepatocytes is not completely understood. In this study, using primary cultured rat hepatocytes, we have shown that interleukin-6 down-regulates expressions of the aldolase B and albumin genes in a dose- and time-dependent manner. We examined whether the decrease in aldolase B and albumin mRNA expressions by interleukin-6 reflected transcriptional down-regulation or stability of the mRNA. Actinomycin D and cycloheximide did not affect the interleukin-6-mediated decrease in the expressions of both genes. These results suggest that the decreased expressions of both genes induced by interleukin-6 is controlled at the transcriptional level, and that it is due neither to increased degradation of mRNA nor to synthesis of new proteins. Protein kinases play a fundamental role in the intracellular signal transduction. To examine the interleukin-6 signal pathway(s) leading to the decrease of aldolase B and albumin mRNA expressions, we tested various kinds of protein kinase inhibitors in this system. Herbimycin A, an inhibitor of tyrosine kinase(s), prevented the decrease in the expression of aldolase B and albumin mRNAs by interleukin-6. H-7, an inhibitor of protein kinase C, prevented the decrease in the expression of albumin mRNA by interleukin-6, but did not induce recovery of that of aldolase B mRNA. These results suggest that a tyrosine kinase(s) or a herbimycin A-sensitive kinase(s) constitutes a common pathway for interleukin-6-mediated reduction of aldolase B and albumin mRNA expressions and that distinct pathways exist for the modes of expression of the two mRNAs.

Cell adhesion to substratum and activation of tyrosine kinases are essentially required for G1/S phase transition in BALB/c 3T3 fibroblasts

Cell adhesion to substratum and activation of tyrosine kinases are essential for the progression of cell cycle through G1 phase in mammalian cells. The kinetic studies of mouse BALB/c 3T3 fibroblasts showed that serum was no longer required for the progression of G1/S phase transition. In contrast, cell adhesion was essentially required in late G1 phase, especially at the period of G1/S transition. Among the kinase inhibitors used to elucidate the signal transduction caused by cell adhesion, tyrosine kinase inhibitors, genistein and herbimycin A, blocked the G1/S transition most effectively when cells were exposed to the inhibitors at the period of G1/S transition. Cell adhesion was not critically required for cells to undergo DNA synthesis once they had passed the G1/S boundary, and the effects of tyrosine kinase inhibitors on the progression of S phase were also not critical. The expressions of histone H2B and dihydrofolate reductase (DHFR) genes (S phase specific genes) and also the transcription factor E2F-1 gene (an activator of DHFR gene) were suppressed when cells were cultured without adhesion or exposed to the tyrosine kinase inhibitors. These results suggest that cell adhesion to substratum plays an important role in the G1/S phase transition of mouse BALB/c 3T3 fibroblasts through the activation of tyrosine kinases other than growth factor receptor-tyrosine kinases.

Differential regulation of liver-specific and ubiquitously-expressed genes in primary rat hepatocytes by the extracellular matrix.

Primary rat hepatocytes were cultured with an extracellular matrix (ECM) overlay, in order to investigate the effect of an ECM on gene expression in hepatocytes. When hepatocytes, isolated by the collagenase-perfusion method, were cultured on type I collagen-coated dishes, the mRNA levels of liver-specific genes (aldolase B, tyrosine aminotransferase and albumin) decreased continuously, while those of ubiquitously-expressed genes (glyceraldehyde 3-phosphate dehydrogenase and β-actin) increased. When a dilute ECM derived from the Engelbreth-Holm-Swarm mouse sarcoma (an EHS gel) was added to the above hepatocytes 3 days after plating, the mRNA levels of liver-specific genes increased, while those of ubiquitously-expressed genes decreased. The effects of a rat liver biomatrix (a physiological ECM for rat hepatocytes) on gene expression in primary hepatocytes were similar to those of the EHS gel. A nuclear run-on assay, and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole or actinomycin D treatments revealed that the transcriptional rates of liver-specific genes were enhanced by the EHS gel overlay, while the apparent stability of the corresponding mRNAs were unchanged. In contrast, the transcriptional rates of ubiquitously-expressed genes were not greatly affected by an EHS gel overlay, while the apparent stability of their mRNAs were decreased. These data suggest that the ECM plays an important role in the maintenance of the differentiated characteristics of primary hepatocytes by inducing the transcription of liver-specific genes and, also, by destabilizing the mRNAs of ubiquitously-expressed genes.

Immunological quantitation of tyrosinase from wild-type and albino mutant mice.

The relationship between gene dosage, enzyme activity, and level of immunologically cross‐reacting material (CRM) was examined in mammalian tyrosinase (EC 1.14.18.1) by rocket immunoelectrophoresis. Skin extracts from mice heterozygous (C/c) and homozygous (c/c) for the albino locus contain 46% and 0% of CRM, respectively, as compared with wild‐type (C/C) animals. Enzyme activity and CRM level were directly proportional in these genotypes, suggesting that the albino locus controls the quantity of tyrosinase produced in melanocytes.

Regulation of Gene Expression by Changes in Cell Adhesion

It is generally accepted that the interaction of cells with their extracellular environment is crucial for their coordinated functions and homeostasis. Differentiation and proliferation are highly coordinated events and represent the most basic behavior of cells. Biological signals to control cellular differentiation and proliferation are initiated by extracellular molecules, which can be divided into two classes based on their solubility. The soluble class includes hormones, growth factors, and cytokines, which circulate in the whole body or diffuse locally and bind to the specific receptors of target organs. The soluble factors are major and potent regulators, and their concentration in serum is finely controlled by integrated biological systems. The insoluble class is represented by (1) cell-cell interaction with adjacent cells and (2) cell adhesion to the extracellular matrix (ECM) (Gumbiner 1996). The insoluble factors are static regulators in comparison with the soluble factors, but have been found to play a critical role in the control of proliferation, differentiation, and morphogenesis.