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Sulforaphane (SFN), an active compound in cruciferous vegetables, has been characterized by its antiproliferative capacity. We investigated the role and molecular mechanism through which SFN regulates proliferation and self-renewal of lung cancer stem cells. CD133+ cells were isolated with MACs from lung cancer A549 and H460 cells. In this study, we found that SFN inhibited the proliferation of lung cancer cells and self-renewal of lung cancer stem cells simultaneously. Meanwhile, the mRNA and protein expressions of Shh, Smo, Gli1 and PHC3 were highly activated in CD133+ lung cancer cells. Compared with siRNA-control group, Knock-down of Shh inhibited proliferation of CD133+ lung cancer cells, and decreased the protein expression of PHC3 in CD133+ lung cancer cells. Knock-down of PHC3 also affected the proliferation and decreased the Shh expression level in CD133+ lung cancer cells. In addition, SFN inhibited the activities of Shh, Smo, Gli1 and PHC3 in CD133+ lung cancer cells. Furthermore, the inhibitory effect of SFN on the proliferation of siRNA-Shh and siRNA-PHC3 cells was weaker than that on the proliferation of siRNA-control cells. Sonic Hedgehog signaling pathway might undergo a cross-talk with PHC3 in self-renewal of lung cancer stem cells. SFN might be an effective new drug which could inhibit self-renewal of lung cancer stem cells through the modulation of Sonic Hedgehog signaling pathways and PHC3. This study could provide a novel way to improve therapeutic efficacy for lung cancer stem cells.

Polycomb Group (PcG) proteins silence critical developmental genes and modulate cell proliferation. Using the Drosophila melanogaster eye as a model system, we show that cells with mutations in the gene locus (ph) that encodes the PcG protein Polyhomeotic (PH) overproliferate and lose both the ability to differentiate and their normal polarity. They invade the neighboring tissues and, when combined with an activated form of the Ras proto-oncogene, they trigger the formation of metastases. PcG proteins bind to multiple genes in the Notch pathway and control their transcription as well as Notch signaling. The massive cell-autonomous overproliferation of ph mutant cell clones can be rescued by ectopic expression of a dominant negative form of Notch or by RNA interference (RNAi)-mediated repression of Notch. Conversely, overexpression of ph induces a small-eye phenotype that is rescued by activation of Notch signaling. These data show that ph is a tumor suppressor locus that controls cellular proliferation by silencing multiple Notch signaling components.

A goal of modern biology is to identify the physical interactions that define ‘functional modules’1 of proteins that govern biological processes. One essential regulatory process is the maintenance of master regulatory genes, such as homeotic genes, in an appropriate ‘on’ or ‘off’ state for the lifetime of an organism. The Polycomb group (PcG) of genes maintain a repressed transcriptional state, and PcG proteins form large multiprotein complexes2,3, but these complexes have not been described owing to inherent difficulties in purification. We previously fractionated a major PcG complex, PRC1, to 20–50% homogeneity from Drosophila embryos. Here, we identify 30 proteins in these preparations, then further fractionate the preparation and use western analyses to validate unanticipated connections. We show that the known PcG proteins Polycomb, Posterior sex combs, Polyhomeotic and dRING1 exist in robust association with the sequence-specific DNA-binding factor Zeste and with numerous TBP (TATA-binding-protein)-associated factors that are components of general transcription factor TFIID (dTAFIIs). Thus, in fly embryos, there is a direct physical connection between proteins that bind to specific regulatory sequences, PcG proteins, and proteins of the general transcription machinery.

polyhomeotic (ph) is a complex locus in Drosophila defined by two genetic units. Two mutational events are necessary to obtain the null lethal phenotype. Molecular analysis has shown that the ph locus contains two transcriptional units coding for two very similar proteins. Although a strong argument in favor of a strict correlation between the genetic and molecular units can be constructed, there is no direct evidence for the hypothesis. Here, we show for all cases with detectable molecular defects that X-ray-induced generation of an amorphic allele from a pre-existing X-ray-induced hypomorphic allele with a lesion limited to one unit invariably involves a rearrangement in the other unit. This result proves that each genetic unit corresponds to one transcription unit.

In Drosophila the Polycomb group (Pc-G) proteins are responsible for the stable and heritable silencing of genes. The Pc-G apparently uses heterochromatin-like mechanisms to transcriptionally inactivate developmental regulators such as the homeotic genes. The Polycomb (Pc) protein is part of a large multimeric complex composed of other members of the Pc-G. We have identified functionally relevant domains of the Pc protein by sequencing different Pc alleles. Additionally, using a Pc-beta gal fusion protein with deleted internal histidine repeats, we found that this mutant protein cannot bind to four particular target loci, but otherwise does not change the remaining overall binding pattern. We show that, in contrast to the dotted subnuclear localization of the wild-type protein, the nuclear distribution of mutant proteins becomes homogeneous. Surprisingly, in Pc mutants the polyhomeotic protein, another member of the Pc-G, is also redistributed in the nucleus. Our results indicate that the appropriate subnuclear localization of the two proteins is critical for the silencing function of the Pc-G complex.

THE bmi-1 proto-oncogene can be activated by Moloney murine leukaemia proviral insertions in Eµ-myc transgenic mice1,2. It encodes a highly conserved nuclear protein of 324 amino acids which belongs to a family of proteins containing a putative new zinc-finger. Another closely related member of this family is the mouse protein Mel-18 (ref. 3). Here we report on the cloning and characterization of a homologous gene (D-bmi) from Drosophila melanogaster. Our analysis indicates that distinct domains of the mouse Bmi-1 protein, including the putative zinc-finger motif, are highly conserved within the much larger D-Bmi protein. Chromosomal localization and sequence comparison reveal that D-bmi is identical to Posterior Sex Combs (Psc)4-6 and indicate that the conserved domains between mouse bmi and Psc are also conserved within Suppressor-2 of Zeste (Su(z)2)6-8

The DNA-binding ETS domain defines the Ets familyof transcription factors. A subset of this family, in-cluding Ets-1, Ets-2, GABPα, Erg, Fli-1 and Tel, alsoshare a conserved Pointed (PNT) or SAM domain thatmay functionin oligomerization,MAP kinase dockingor association with other regulatory proteins. The so-lution structure of the PNT/SAM domain from Ets-1(PDB: 1qbv; Slupsky et al., 1998a) has an architec-ture of a core four α-helical bundle interfaced withan additional N-terminal helix. In contrast, the crystalstructure of the homologous domain from Tel (1ji7;Kim et al., 2001) shows only the four core helices, asalso seen in the structures of SAM domains from non-Ets family memberssuch as p73 (1cok), polyhomeotic(1kw4) and the Ephrin receptors (1b4f, 1sgg, 1b0x).The additional N-terminal helix present in Ets-1 is anintegral part of the structured PNT/SAM domain asevident by

A new locus in Drosophila melanogaster that is required for the correct expression of segmental identity has been discovered. The new locus, termed polyhomeotic (ph), is X-linked and maps cytologically to bands 2D2-3. Homozygous ph flies have homeotic transformations similar to those of known dominant gain of function mutants in the Antennapedia and bithorax complexes (ANT-C, BX-C), and in addition show loss of the humerus. ph interacts with three other similar mutations: Polycomb (Pc), Polycomblike (Pcl), and extra sex comb (esc), and acts as a dominant enhancer of Pc. The expression of ph depends on the ANT-C and BX-C dosage. ph has no embryonic phenotype, but temperature shift studies on ph2 show that the ph+ product is required during embryogenesis and larval development. We propose that ph mutants in some way disrupt the normal expression of the ANT-C and BX-C, and, therefore, that ph+ is needed for maintenance of segmental identity.

The bithorax gene complex in Drosophila contains a minimum of eight genes that seem to code for substances controlling levels of thoracic and abdominal development. The state of repression of at least four of these genes is controlled by cis-regulatory elements and a separate locus (Polycomb) seems to code for a repressor of the complex. The wild-type and mutant segmentation patterns are consistent with an antero-posterior gradient in repressor concentration along the embryo and a proximo-distal gradient along the chromosome in the affinities for repressor of each gene's cis-regulatory element.

The morphological diversity among the body segments of Drosophila, as displayed in the pattern of the larval cuticle1, arises during embryogenesis by the selective expression of homoeotic genes. For example, the bithorax complex (BX-C) confers abdominal development on the segments in the posterior half of the embryo which otherwise would develop into thoracic segments2. Mutations in trans-regulatory genes such as Polycomb (Pc)2 or extra sex combs (esc)3 cause indiscriminate expression of the BX-C genes, which leads to all body segments being abdominal. I report here on mutations in four other trans-regulatory genes, members of a novel class of more than 20 genes controlling the spatial expression of BX-C genes. In contrast to Pc and esc mutations, only partial transformation into posterior abdominal development is observed in mutant embryos. However, embryos mutant for two or more of these genes show strong homoeotic transformation of all body segments similar to, or stronger than, that seen in Pc and esc embryos, indicating that these genes act synergistically in normal development to control the spatial expression of the BX-C genes.