Yoshiaki Monden

Effect of genistein on topoisomerase activity and on the growth of [Val 12]Ha-ras-transformed NIH 3T3 cells

Genistein inhibited topoisomerase II and I; it increased the enzyme-DNA complex in L1210 cells at 1 micrograms/ml, and interfered with pBR322 DNA relaxation by the enzymes. To test the role of topoisomerase in the transformation by oncogenes, the effect of genistein on the transformation of NIH 3T3 cells by transfection with [Val 12]Ha-ras was compared with that of N-alpha-tosyl-L-lysyl-chloromethyl ketone (TLCK), since genistein inhibits tyrosine kinase as well as TLCK. Genistein reduced the number of foci of the transformed cells, and suppressed selectively the growth of ras-transformed NIH 3T3 cells but not normal NIH 3T3 cells. In contrast, TLCK did not affect the transformation. It inhibited the growth of the normal cells but not the transformed cells.

Malolactomycin D, a potent inhibitor of transcription controlled by the Ras responsive element, inhibits Ras-mediated transformation activity with suppression of MMP-1 and MMP-9 in NIH3T3 cells.

To search for anti-cancer agents, a screening system for Ras signal inhibitors was developed using a NIH3T3 cell line with an introduced reporter gene which is controlled by the Ras-responsive element (RRE). With this screening system, malolactomycin D was identified as a selective inhibitor of transcription from the RRE. This compound was found to preferentially inhibit the anchorage-independent growth rather than the anchorage-dependent growth of Ras-transformed NIH3T3 cells. The expression of matrix metalloproteinases MMP-1 and MMP-9, which have RRE in their promoters, were reduced by treatment with malolactomycin D at the translational and transcriptional levels. Analysis of the activity of mitogen-activated protein (MAP) kinases, which play important roles in transduction of the Ras signal, showed that malolactomycin D inhibits the activation of p38 MAP kinase and Jun N-terminal-kinase (JNK) but not extracellular signal-regulated kinase 1 or 2 (ERK1 or 2). These findings suggest that by inhibiting the pathway that leads to the activation of p38 MAP kinase and JNK, malolactomycin D suppresses the expression of MMPs. Since MMPs play important roles in metastasis and maintenance of the microenvironment of tumor cells, both of which facilitate tumor growth, the inhibition of MMPs by malolactomycin D is believed to contribute to its ability to inhibit Ras-mediated tumorigenesis.

Azatyrosine: Mechanism of Action for Conversion of Transformed Phenotype to Normal

Abstract: Azatyrosine [L‐β‐(5‐hydroxy‐2‐pyridyl)‐alanine] has the unique property of converting ras‐ or c‐erbB‐2 transformed phenotype to normal. The administration of azatyrosine also inhibits tumor formation in transgenic mice harboring the normal human c‐Ha‐ras which is mutated during treatment with various chemical carcinogens. To elucidate the molecular mechanism, we investigated how azatyrosine functions and what are its major targets. Azatyrosine functions downstream of ras; azatyrosine does not alter either the level of GTP‐bound Ras or the total amount of Ras. Instead, azatyrosine inhibits the activation of c‐Raf‐1 kinase by oncogenic c‐ErbB‐2, resulting in inactivation of AP1. It is interesting that azatyrosine also restores the expression of the rhoB gene, the product of which regulates the formation of actin stress fibers. Azatyrosine is incorporated into cellular proteins to replace tyrosine. Several experiments indicate that replacement of tyrosine is likely to be a cause for its conversion of ransformed phenotype to normal. To prove this hypothesis, we are attempting to develop a mutant of tyrosyl‐tRNA synthetase that, unlike wild type, can aminoacylate azatyrosine more efficiently than can tyrosine.

Failure to Detect Mutations in the Retinoblastoma Protein‐binding Domain of the Transcription Factor E2F‐1 in Human Cancers

The functions of the transcription factor E2F‐1 are regulated by the RB protein through the RB‐binding domain of E2F‐1 and this factor is considered to be an important molecule that functions downstream of the RB protein. In order to determine whether E2F‐1 that cannot bind to RB might be associated with various human cancers, we searched for mutations in the RB‐binding domain of E2F‐1 using samples of DNA from various clinical specimens obtained from 406 cancer patients (with lung, pancreatic, stomach, colon, esophageal, and hepatic cancers) by analysis of polymerase chain reaction‐mediated single‐strand conformational polymorphism. No mutations or deletions were detected in genes for E2F‐1 from any of the tumor tissues examined. These results suggest that a mutation or deletion in E2F‐1 that might affect binding of the RB protein is not involved in human cancers.