The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) and (3;21)(q26;q22) translocations associated with myelogenous leukemias and encodes a DNA binding protein. From the AML1 gene, two representative forms of proteins, AML1a and AML1b, are produced by alternative splicing. Both forms have a DNA binding domain but, unlike AML1b, AML1a lacks a putative transcriptional activation domain. Here we demonstrate that overexpressed AML1a totally suppresses granulocytic differentiation and stimulates cell proliferation in 32Dcl3 murine myeloid cells treated with granulocyte colony‐stimulating factor. These effects of AML1a were canceled by the concomitant overexpression of AML1b. Such biological phenomena could be explained by our observations that (i) AML1a, which on its own has no effects as a transcriptional regulator, dominantly suppresses transcriptional activation by AML1b, and (ii) AML1a exhibits the higher affinity for DNA binding compared with AML1b. These antagonistic actions could be important in leukemogenesis and/or myeloid cell differentiation because more than half of myelogenous leukemia patients showed an increase in the relative amounts of AML1a.

UMIN000001396.

ABSTRACT:A novel and convenient method was established for the prediction of in vivo metabolic clearance in human liver. The present method applied the in vitro-in vivo extrapolation paradigm previously established in rats to the in vitro data obtained from cryopreserved human hepatocytes. Predicted hepatic availability and clearance were compared with the reported oral bioavailability and plasma clearance in humans for 14 clinically used drugs (naloxone, buspirone, verapamil, lidocaine, imipramine, metoprolol, timolol, antipyrine, diazepam, quinidine, caffeine, propranolol, diclofenac, and phenacetin). A large interindividual variation was observed in the intrinsic metabolic clearance among separate cryopreserved preparations from different subjects. The prediction generally resulted in a marked underestimation when the biologically based scaling (cells/kg) from pooled preparation of two selected lots, which were 3-to 4-fold larger than the biologically based scaling factor. These data suggested that the calibration of inherent interindividual variation of metabolic activities among different cryopreserved preparations of human hepatocytes to obtain the empirical scaling factor, which is applicable only to the preparation used, was an essential step for more reliable and quantitative prediction of in vivo metabolic activity in humans.Hepatic clearance for the metabolism of compounds kinetically consists of two major determinants: intrinsic (metabolic) clearance of the unbound compound and unbound fraction of compound in the blood (or plasma when corrected by the blood-to-plasma partition). Generally, the intrinsic clearance for the unbound compound is measured in vitro by the incubation of isolated hepatocytes or subcellular fractions such as S-9 and microsomes in the protein-free medium. The in vitro metabolic parameters thus obtained are extrapolated by using anatomical parameters such as cell numbers and protein content in the intact liver for the prediction of in vivo metabolic activity (Houston and Carlile, 1997;Iwatsubo et al., 1997;Obach, 1999). Separate experiments necessarily are further carried out to measure the unbound fraction in the plasma. Many technical problems including adsorption of compounds to the apparatus during the equilibrium dialysis and ultra-filtration often hamper the accuracy of the evaluated values (Bertilsson et al., 1979;Desoye, 1988). To improve the accuracy and avoid complexity for predicting in vivo metabolic clearance from in vitro experiments, we have recently developed a novel and convenient in vitro method for predicting in vivo metabolic clearance by using freshly isolated rat hepatocytes suspended in rat serum (Shibata et al., 2000). Oral bioavailability and hepatic clearance for 16 widely used compounds were well predicted directly from the in vitro metabolic clearance values obtained from a single incubation without separate evaluation of unbound fraction in the plasma. The purposes of the present study were to 1) determine whether the same methodology was applicable ...

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The molecular chaperone HSP90 plays a crucial role in cancer cell growth and survival by stabilizing cancer-related proteins. A number of HSP90 inhibitors have been developed clinically for cancer therapy; however, potential off-target and/or HSP90-related toxicities have proved problematic. The 4-(1H-pyrazolo [3,4-b]pyridine-1-yl)benzamide TAS-116 is a selective inhibitor of cytosolic HSP90a and b that does not inhibit HSP90 paralogs such as endoplasmic reticulum GRP94 or mitochondrial TRAP1. Oral administration of TAS-116 led to tumor shrinkage in human tumor xenograft mouse models accompanied by depletion of multiple HSP90 clients, demonstrating that the inhibition of HSP90a and b alone was sufficient to exert antitumor activity in certain tumor models. One of the most notable HSP90-related adverse events universally observed to differing degrees in the clinical setting is visual disturbance. A two-week administration of the isoxazole resorcinol NVP-AUY922, an HSP90 inhibitor, caused marked degeneration and disarrangement of the outer nuclear layer of the retina and induced photoreceptor cell death in rats. In contrast, TAS-116 did not produce detectable photoreceptor injury in rats, probably due to its lower distribution in retinal tissue. Importantly, in a rat model, the antitumor activity of TAS-116 was accompanied by a higher distribution of the compound in subcutaneously xenografted NCI-H1975 non-small cell lung carcinoma tumors than in retina. Moreover, TAS-116 showed activity against orthotopically transplanted NCI-H1975 lung tumors. Together, these data suggest that TAS-116 has a potential to maximize antitumor activity while minimizing adverse effects such as visual disturbances that are observed with other compounds of this class.

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