The removal of intervening sequences from transcripts is catalyzed by the spliceosome, a multicomponent complex that assembles on the newly synthesized pre-mRNA. Pre-mRNA translation in the cytoplasm leads to the generation of aberrant proteins that are potentially harmful. Therefore, tight control to prevent undesired pre-mRNA export from the nucleus and its subsequent translation is an essential requirement for reliable gene expression. Here, we show that the natural product FR901464 (1) and its methylated derivative, spliceostatin A (2), inhibit in vitro splicing and promote pre-mRNA accumulation by binding to SF3b, a subcomplex of the U2 small nuclear ribonucleoprotein in the spliceosome. Importantly, treatment of cells with these compounds resulted in leakage of pre-mRNA to the cytoplasm, where it was translated. Knockdown of SF3b by small interfering RNA induced phenotypes similar to those seen with spliceostatin A treatment. Thus, the inhibition of pre-mRNA splicing during early steps involving SF3b allows unspliced mRNA leakage and translation.

Cyclin D1 binds to the Cdk4 and Cdk6 to form a pRB kinase. Upon phosphorylation, pRB loses its repressive activity for the E2F transcription factor, which then activates transcription of several genes required for the transition from the G1-to S-phase and for DNA replication. The cyclin D1 gene is rearranged and overexpressed in centrocytic lymphomas and parathyroid tumors and it is amplified and/or overexpressed in a major fraction of human tumors of various types of cancer. Ectopic overexpression of cyclin D1 in fibroblast cultures shortens the G1 phase of the cell cycle. Furthermore, it has been demonstrated that introduction of an antisense cyclin D1 into a human carcinoma cell line, in which the cyclin D1 gene is amplified and overexpressed, causes reversion of the malignant phenotype. Thus, increased expression of cyclin D1 can play a critical role in tumor development and in maintenance of the malignant phenotype. However, it is insufficient to confer transformed properties on primary or established fibroblasts. In this review, we summarize the role of cyclin D1 on tumor development and malignant transformation. In addition, our chemical biology study to understand the regulatory mechanism of cyclin D1 transcription is also reviewed. (Cancer Sci 2007; 98: 629 -635) I n mammalian cells, the progression of replicating cells through the cell cycle is controlled by the sequential formation, activation, and subsequent inactivation of a series of specific cyclindependent kinase (CDK) complexes. The second regulatory mechanism involves the binding of specific inhibitory proteins (p21WAF1, p27KIP1, p57KIP2) to cyclin-CDK complexes or the binding of specific inhibitory proteins directly to CDK4 (p15, p16INK1, p18, and p19).(1−3) There is now abundant evidence that disturbances in specific cyclins, CDKs, or the above-mentioned inhibitory proteins play an important role in several types of human cancer.(1−3) The most frequent abnormalities relate to cyclin D1. The cyclin D1 gene encodes a regulatory subunit of the cdk4 and cdk6 holoenzyme complex, which phosphorylates and deactivates the tumor suppressor protein pRB.(3) The phophorylation of pRB results in its inactivation and the release of E2Fs that have been sequestered by the unphosphorylated (active) form of pRB. Once liberated by pRB inactivation, E2Fs then proceed to activate genes that are essential for advances into the late G1 and S phases. Consistent with its growth-promoting role, cyclin D1 can act as an oncogene. Indeed, rearrangement, amplification, and/or increased expression of the cyclin D1 gene and overexpression of its mRNA have been reported in several types of human cancer, including human parathyroid adenomas, B cell lymphomas, breast, colon, lung, bladder and liver cancers, and squamous carcinomas of the esophagus, head and neck. (2,5,6) The expression of cyclin D1 mRNA and protein peaks during mid-G1 when growth factor-deprived cells are restimulated to enter the cell cycle. (7,8) Inhibition of cyclin D1 expression either by antisense methodo...

The IRE1α-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Therefore, the availability of small-molecule inhibitors targeting this pathway would offer a new chemotherapeutic strategy for MM. Here, we screened small-molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) activation. Furthermore, although toyocamycin was unable to inhibit IRE1α phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Toyocamycin inhibited not only ER stress-induced but also constitutive activation of XBP1 expression in MM lines as well as primary samples from patients. It showed synergistic effects with bortezomib, and induced apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM. Taken together, our results suggest toyocamycin as a lead compound for developing anti-MM therapy and XBP1 as an appropriate molecular target for anti-MM therapy.