Purpose: Bendamustine has shown clinical activity in patients with disease refractory to conventional alkylator chemotherapy. The purpose of this study was to characterize the mechanisms of action of bendamustine and to compare it with structurally related compounds. Experimental Design: Bendamustine was profiled in the National Cancer Institute in vitro antitumor screen. Microarray-based gene expression profiling, real-time PCR, immunoblot, cell cycle, and functional DNA damage repair analyses were used to characterize response to bendamustine and compare it with chlorambucil and phosphoramide mustard. Results: Bendamustine displays a distinct pattern of activity unrelated to other DNA-alkylating agents. Its mechanisms of action include activation of DNA-damage stress response and apoptosis, inhibition of mitotic checkpoints, and induction of mitotic catastrophe. In addition, unlike other alkylators, bendamustine activates a base excision DNA repair pathway rather than an alkyltransferase DNA repair mechanism. Conclusion: These results suggest that bendamustine possesses mechanistic features that differentiate it from other alkylating agents and may contribute to its distinct clinical efficacy profile.

Background: The draft genome sequence of the ascidian Ciona intestinalis, along with associated gene models, has been a valuable research resource. However, recently accumulated expressed sequence tag (EST)/cDNA data have revealed numerous inconsistencies with the gene models due in part to intrinsic limitations in gene prediction programs and in part to the fragmented nature of the assembly.

The ascidian tadpole represents the most simplified chordate body plan. It contains a notochord composed of just 40 cells, but as in vertebrates Brachyury is essential for notochord differentiation. Here, we show that the misexpression of the Brachyury gene (Ci-Bra) of Ciona intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra misexpression. Of 501 independent cDNA clones that were surveyed, 38 were specifically expressed in notochord cells. These potential CiBra downstream genes appear to encode a broad spectrum of divergent proteins associated with notochord formation. Received March 22, 1999; revised version accepted May 3, 1999. Brachyury encodes a sequence-specific activator that contains a T-box DNA-binding domain (Herrmann et al. 1990;Kispert et al. 1995;Conlon et al. 1996). In vertebrates, Brachyury is initially expressed throughout the presumptive mesoderm, and during later stages the expression pattern is gradually restricted to the developing notochord and tailbud. Brachyury notochord differentiation is essential in all vertebrates that have been studied, including mice, frogs, and zebrafish (for review, see Herrmann and Kispert 1994;Smith 1997;Papaioannou and Silver 1998).Brachyury is expressed exclusively in the notochord precursor cells of two divergent ascidians, Halocynthia roretzi (Yasuo and Satoh 1993) and Ciona intestinalis (Corbo et al. 1997a). The spatial and temporal patterns of the gene expression coincide with the clonal restriction of the notochord lineages. In H. roretzi, notochord formation is induced at the 32-cell stage by signals emanating from the adjacent endoderm (Nakatani and Nishida 1994). Overexpression of the Halocynthia Brachyury gene (As-T) via RNA injection results in notochord formation without a requirement for the inductive event at the 32-cell stage (Yasuo and Satoh 1998). In addition, misexpression of As-T also causes transformation of endoderm and neuronal lineages into notochord (Yasuo and Satoh 1998). These results indicate that the ascidian Brachyury gene is a critical determinant of the notochord. Here we report that the misexpression of the Brachyury gene (Ci-Bra) of C. intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra overexpression. We isolated and characterized 38 different notochord-specific genes that may include potential targets of the ascidian Brachyury. Results and DiscussionThe fork head/HNF-3␤ gene of C. intestinalis (Ci-fkh) is expressed in the endoderm, endodermal strand, notochord, and ventral ependymal cells of the neural tube (Corbo et al. 1997b). A 2.6-kb genomic DNA fragment from the 5Ј-flanking region of Ci-fkh is sufficient to direct the expression of a lacZ reporter gene in these tissues after electroporation into one-cell embryos (Fig. 1A). The Ci-Bra gene was misexpr...

The swimming larvae of many marine animals identify a location on the sea floor to undergo metamorphosis based on the presence of specific bacteria. Although this microbe–animal interaction is critical for the life cycles of diverse marine animals, what types of biochemical cues from bacteria that induce metamorphosis has been a mystery. Metamorphosis of larvae of the tubeworm Hydroides elegans is induced by arrays of phage tail-like contractile injection systems, which are released by the bacterium Pseudoalteromonas luteoviolacea. Here we identify the novel effector protein Mif1. By cryo-electron tomography imaging and functional assays, we observe Mif1 as cargo inside the tube lumen of the contractile injection system and show that the mif1 gene is required for inducing metamorphosis. Purified Mif1 is sufficient for triggering metamorphosis when electroporated into tubeworm larvae. Our results indicate that the delivery of protein effectors by contractile injection systems may orchestrate microbe–animal interactions in diverse contexts.

SUMMARYThe nervous system-enriched microRNA miR-124 is necessary for proper nervous system development, although the mechanism remains poorly understood. Here, through a comprehensive analysis of miR-124 and its gene targets, we demonstrate that, in the chordate ascidian Ciona intestinalis, miR-124 plays an extensive role in promoting nervous system development. We discovered that feedback interaction between miR-124 and Notch signaling regulates the epidermal-peripheral nervous system (PNS) fate choice in tail midline cells. Notch signaling silences miR-124 in epidermal midline cells, whereas in PNS midline cells miR-124 silences Notch, Neuralized and all three Ciona Hairy/Enhancer-of-Split genes. Furthermore, ectopic expression of miR-124 is sufficient to convert epidermal midline cells into PNS neurons, consistent with a role in modulating Notch signaling. More broadly, genome-wide target extraction with validation using an in vivo tissue-specific sensor assay indicates that miR-124 shapes neuronal progenitor fields by downregulating non-neural genes, notably the muscle specifier Macho-1 and 50 Brachyuryregulated notochord genes, as well as several anti-neural factors including SCP1 and PTBP1. 3ЈUTR conservation analysis reveals that miR-124 targeting of SCP1 is likely to have arisen as a shared, derived trait in the vertebrate/tunicate ancestor and targeting of PTBP1 is conserved among bilaterians except for ecdysozoans, while extensive Notch pathway targeting appears to be Ciona specific. Altogether, our results provide a comprehensive insight into the specific mechanisms by which miR-124 promotes neuronal development.