The form of RNA polymerase II (RNAPII) engaged in transcriptional elongation was isolated. Elongating RNAPII was associated with a novel multisubunit complex, termed elongator, whose stable interaction was dependent on a hyperphosphorylated state of the RNAPII carboxy-terminal domain (CTD). A free form of elongator was also isolated, demonstrating the discrete nature of the complex, and free elongator could bind directly to RNAPII. The gene encoding the largest subunit of elongator, ELP1, was cloned. Phenotypes of yeast elp1 delta cells demonstrated an involvement of elongator in transcriptional elongation as well as activation in vivo. Our data indicate that the transition from transcriptional initiation to elongation involves an exchange of the multiprotein mediator complex for elongator in a reaction coupled to CTD hyperphosphorylation.

The elongator complex is a major component of the RNA polymerase II (RNAPII) holoenzyme responsible for transcriptional elongation in yeast. Here we identify Elp3, the 60-kilodalton subunit of elongator/RNAPII holoenzyme, as a highly conserved histone acetyltransferase (HAT) capable of acetylating core histones in vitro. In vivo, ELP3 gene deletion confers typical elp phenotypes such as slow growth adaptation, slow gene activation, and temperature sensitivity. These results suggest a role for a novel, tightly RNAPII-associated HAT in transcription of DNA packaged in chromatin.

Human Elongator complex was purified to virtual homogeneity from HeLa cell extracts. The purified factor can exist in two forms: a six-subunit complex, holo-Elongator, which has histone acetyltransferase activity directed against histone H3 and H4, and a three-subunit core form, which does not have histone acetyltransferase activity despite containing the catalytic Elp3 subunit. Elongator is a component of early elongation complexes formed in HeLa nuclear extracts and can interact directly with RNA polymerase II in solution. Several human homologues of the yeast Elongator subunits were identified as subunits of the human Elongator complex, including StIP1 (STAT-interacting protein 1) and IKAP (IKK complex-associated protein). Mutations in IKAP can result in the severe human disorder familial dysautonomia, raising the possibility that this disease might be due to compromised Elongator function and therefore could be a transcription disorder.

Despite recent progress in its treatment, multiple myeloma (MM) remains incurable, thus necessitating identification of novel anti-MM agents. We report that the marine-derived cyclodepsipeptide Aplidin exhibits, at clinically achievable concentrations, potent in vitro activity against primary MM tumor cells and a broad spectrum of human MM cell lines, including cells resistant to conventional (e.g., dexamethasone, alkylating agents, and anthracyclines) or novel (e.g., thalidomide and bortezomib) anti-MM agents. Aplidin is active against MM cells in the presence of proliferative/antiapoptotic cytokines or bone marrow stromal cells and has additive or synergistic effects with some of the established anti-MM agents. Mechanistically, a short in vitro exposure to Aplidin induces MM cell death, which involves activation of p38 and c-jun NH 2 -terminal kinase signaling, Fas/CD95 translocation to lipid rafts, and caspase activation. The anti-MM effect of Aplidin is associated with suppression of a constellation of proliferative/antiapoptotic genes (e.g., MYC, MYBL2, BUB1, MCM2, MCM4, MCM5, and survivin) and up-regulation of several potential regulators of apoptosis (including c-JUN, TRAIL, CASP9, and Smac). Aplidin exhibited in vivo anti-MM activity in a mouse xenograft model. The profile of the anti-MM activity of Aplidin in our preclinical models provided the framework for its clinical testing in MM, which has already provided favorable preliminary results. [Cancer Res 2008; 68(13):5216-25]

No abstract

We have analyzed the DNA binding properties of the antitumor agent trabectedin (ET-743, Yondelis) and different analogs, namely, ET-745, lacking the C21-hydroxyl group, and ET-637, ET-594, ET-637-OBu, with modifications at the trabectedin C domain, versus their effects on cell cycle, apoptosis, and gene expression. ET-745 failed to bind DNA, highlighting the importance of the C21-hydroxyl group for DNA binding. Analogs ranked trabectedin >> ET-637 approximately ET-594 > ET-637-OBu >> ET-745 for their DNA binding capacity; ET-637 and ET-594 display very different biological activities. Drugs were clustered in three major groups showing high (trabectedin, ET-637), intermediate (ET-637-OBu), and low (ET-594, ET-745) cytotoxic activity and similar transcriptional profiling responses. C21-hydroxyl-deficient analogs of the above-mentioned compounds showed a dramatic decrease in biological activity. Our data suggest that trabectedin interacts with an additional non-DNA target to raise an effective antitumor response, and that this interaction is favored through trabectedin-DNA complexes.

The isolation of new molecules from marine sources opens the door to their possible therapeutic use against tumors and other pathological conditions. Indeed, we recently defined the cytotoxicity of ES 285, obtained from the clam Mactromeris polynima, and its affects on the cells microfilament but not the microtubule network. Considering the analogy between ES 285 and sphingosine-related lipids, we wondered whether ES 285 might affect the activity of PKC at the intracellular level. While we anticipated that ES 285 might inhibit PKC, it turns out that in contrast it serves to activate PKC at the cellular level. Indeed, like other sphingosine-related lipids, ES 285 induces the phosphorylation of MARCKS. Additionally, we further examined the cytotoxicity of ES 285 to elucidate the molecular mechanisms through which this compound triggers apoptosis. When the influence of ES 285 on "cell death markers" was assessed, it became clear that ES285 activates caspase 3 and 12, and that it modified the phosphorylation of p53. In contrast, ES 285 does not affect other pathways widely implicated in regulating cell survival/apoptosis, such as JNK, Erks or Akt. Thus, these data suggest that ES 285-triggers an atypical cell death program when compared to other sphingosine-dependent apoptosis pathways.

No abstract