Cell growth and differentiation are usually antagonistic. Proteins of the basic helix-loop-helix (bHLH) family bind DNA and play important roles in the differentiation of specific cell types. Id proteins heterodimerize with bHLH transcription factors, blocking their activation of lineage-specific gene expression and thereby inhibiting cellular differentiation. To examine the effect of Id-2 on cell proliferation, we overexpressed Id-2 in the human osteosarcoma cell line U2OS. Id-2 expression in U2OS reduced the serum requirement for growth and stimulated cellular proliferation by shortening the doubling time and increasing the percentage of cells in S phase. We demonstrated that Id-2 expression was able to reverse the inhibition of cellular proliferation and the block in cell cycle progression mediated by the product of the retinoblastoma tumor suppressor gene pRB. This effect was not associated with changes in the state of pRb phosphorylation in transfected cells. In vitro, unphosphorylated pRb from cell lysates specifically bound Id-2 but was not able to bind a mutated form of Id-2 lacking the HLH domain that also did not antagonize the growth arrest by pRb. In vitro-synthesized pRb containing mutations within the E1A/large T-binding pocket did not bind Id-2. However, wild-type pRb was able to bind to a region of Id-2 corresponding to only the HLH domain. In vivo, a physical association between Id-2 and pRb was seen in cross-linked extracts from SAOS-2 cells transfected with Id-2 and pRb. Our data identify a role for Id-2 in the regulation of cellular proliferation and suggest that the interaction between Id-2 and pRb is a molecular pathway over which synchronous changes in growth and differentiation are mediated in vivo.

The phytochemical indole-3-carbinol (I3C), from cruciferous vegetables such as broccoli, has been shown to elicit a potent anti-proliferative response in human breast cancer cell lines. Treatment of the immortalized human mammary epithelial cell line MCF10A with I3C induced a G1 cell cycle arrest, elevated p53 tumor suppressor protein levels and stimulated expression of downstream transcriptional target, p21. I3C treatment also elevated p53 levels in several breast cancer cell lines that express mutant p53. I3C did not arrest MCF10A cells stably transfected with dominant-negative p53, establishing a functional requirement for p53. Cell fractionation and immunolocalization studies revealed a large fraction of stabilized p53 protein in the nucleus of I3C-treated MCF10A cells. With I3C treatment, phosphatidyl-inositol-3-kinase family member ataxia telangiectasia-mutated (ATM) was phosphorylated, as were its substrates p53, CHK2 and BRCA1. Phosphorylation of p53 at the N-terminus has previously been shown to disrupt the interaction between p53 and its ubiquitin ligase, MDM2, and therefore stabilizing p53. Coimmunoprecipitation analysis revealed that I3C reduced by 4-fold the level of MDM2 protein that associated with p53. The p53-MDM2 interaction and absence of p21 production were restored in cells treated with I3C and the ATM inhibitor wortmannin. Significantly, I3C does not increase the number of 53BP1 foci or H2AX phosphorylation, indicating that ATM is activated independent of DNA double-strand breaks. Taken together, our results demonstrate that I3C activates ATM signaling through a novel pathway to stimulate p53 phosphorylation and disruption of the p53-MDM2 interaction, which releases p53 to induce the p21 CDK inhibitor and a G1 cell cycle arrest. ' 2005 Wiley-Liss, Inc.Key words: indole-3-carbinol; breast cancer; p53; MDM2; cell cycle arrest; ATM Indole-3-carbinol (I3C), a phytochemical of Brassica vegetables such as broccoli and cabbage, has been shown to prevent spontaneous and carcinogen-induced mammary tumors.1-3 I3C has a direct anti-proliferative effect in both estrogen-dependent and independent human breast cancer cell lines. 4 In tissue culture, I3C treatment of human adenocarcinoma cells induces a G1 cell cycle arrest that is associated with a decreased expression of cyclindependent kinase 6 (CDK6), and inhibited specific enzymatic activity of CDK2. 4,5 In [ 3 H]I3C-treated MCF-7 cells, the I3C condensation product 3,3 0 -diindolylmethane (DIM) represents 40% of radiolabeled compounds in the nuclear fraction, suggesting that DIM may mediate a subset of the transcriptional responses to I3C. 6 DIM has been shown to decrease CDK2 activity through Sp1-mediated transcriptional upregulation of CDK inhibitor p21. 7In addition to their anti-proliferative effects, treatment with I3C or DIM also causes a p53-independent apoptotic response in cultured breast cancer cells. [8][9][10] I3C was shown to induce apoptosis of the tumorigenic MCF10CA1a cell line, 11 but the mechanism of growth inhibition of the isogen...

Using intracellular lineage tracers to study the main neurogenic lineage (N lineage) of the glossiphoniid leech embryo, we have characterized events leading from continuous columns of segmental founder cells (nf and ns primary blast cells) to discrete, segmentally iterated ganglia. The separation between prospective ganglia was first evident as a fissure between the posterior boundary of nf- and the anterior boundary of ns-derived progeny. We also identified the sublineages of nf-derived cells that contribute parallel stripes of cells to each segment. These stripes of cells project ventrolaterally from the dorsolateral margin of each nascent ganglion to the ventral body wall. The position and orientation of the stripes suggests that they play a role in forming the posterior segmental nerve; they are not coincident with the ganglionic boundary, and they form well after the separation of ganglionic primordia. Previous work has shown that cells in the anterior stripe express the leech engrailed-class gene. Thus, in contrast to the role of cells expressing engrailed in Drosophila, the stripes of N-derived cells expressing an engrailed-class gene in leech do not seem to play a direct role in segmentation or segment polarity.

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Indole-3-carbinol (I3C), a naturally occurring compound found in vegetables of the Brassica genus, such as broccoli and cabbage, is a promising anticancer agent previously shown to induce a G(1) cell-cycle arrest in the cells of human lymph node carcinoma of prostate (LNCaP) through regulation of specific G(1)-acting cell-cycle components. Since the androgen receptor (AR) mediates proliferation and differentiation in the prostate and is expressed in nearly all human prostate cancers, the effects of I3C on AR expression and function were examined in LNCaP cells. Immunoblot and quantitative RT-PCR assays revealed that I3C inhibited the expression of AR protein and mRNA levels within 12 h of indole treatment. I3C downregulated the reporter activity of LNCaP cells transiently transfected with an AR promoter-luciferase plasmid, demonstrating that a unique response to I3C is the inhibition of AR promoter activity. In contrast to I3C, the natural I3C dimerization product 3,3'-diindolylmethane, which acts as an androgen antagonist, had no effect on AR expression. To determine the functional significance of the I3C-inhibited expression of AR, the AR-regulated prostate specific antigen (PSA) was utilized as a downstream indicator. I3C downregulated the expression of PSA transcripts and protein levels and inhibited PSA promoter activity, as well as that of a minimal androgen responsive element containing reporter plasmid. Expression of exogenous AR prevented the I3C disruption of androgen-induced PSA expression. Taken together, our results demonstrate that I3C represses AR expression and responsiveness in LNCaP cells as a part of its antiproliferative mechanism.