The biological functions of nuclear topoisomerase I (Top1) have been difficult to study because knocking out TOP1 is lethal in metazoans. To reveal the functions of human Top1, we have generated stable Top1 small interfering RNA (siRNA) cell lines from colon and breast carcinomas (HCT116-siTop1 and MCF-7-siTop1, respectively). In those clones, Top1 is reduced f5-fold and Top2A compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone ;-H2AX foci associated with replication defects. siTop1 cells also show rDNA and nucleolar alterations and increased nuclear volume. Genome-wide transcription profiling revealed 55 genes with consistent changes in siTop1 cells. Among them, asparagine synthetase (ASNS) expression was reduced in siTop1 cells and in cells with transient Top1 down-regulation. Conversely, Top1 complementation increased ASNS, indicating a causal link between Top1 and ASNS expression. Correspondingly, pharmacologic profiling showed L-asparaginase hypersensitivity in the siTop1 cells. Resistance to camptothecin, indenoisoquinoline, aphidicolin, hydroxyurea, and staurosporine and hypersensitivity to etoposide and actinomycin D show that Top1, in addition to being the target of camptothecins, also regulates DNA replication, rDNA stability, and apoptosis. Overall, our studies show the pleiotropic nature of human Top1 activities. In addition to its classic DNA nicking-closing functions, Top1 plays critical nonclassic roles in genomic stability, gene-specific transcription, and response to various anticancer agents. The reported cell lines and approaches described in this article provide new tools to perform detailed functional analyses related to Top1 function.

Profiling the dynamic interaction of p300 with proximal promoters of human T cells identified a class of genes that rapidly coassemble p300 and RNA polymerase II (pol II) following mitogen stimulation. Several of these p300 targets are immediate early genes, including FOS, implicating a prominent role for p300 in the control of primary genetic responses. The recruitment of p300 and pol II rapidly transitions to the assembly of several elongation factors, including the positive transcriptional elongation factor (P-TEFb), the bromodomain-containing protein (BRD4), and the elongin-like eleven nineteen lysine-rich leukemia protein (ELL). However, transcription at many of these rapidly induced genes is transient, wherein swift departure of P-TEFb, BRD4, and ELL coincides with termination of transcriptional elongation. Unexpectedly, both p300 and pol II remain accumulated or ''bookmarked'' at the proximal promoter long after transcription has terminated, demarking a clear mechanistic separation between the recruitment and elongation phases of transcription in vivo. The bookmarked pol II is depleted of both serine-2 and serine-5 phosphorylation of its C-terminal domain and remains proximally positioned at the promoter for hours. Surprisingly, these p300/pol II bookmarked genes can be readily reactivated, and elongation factors can be reassembled by subsequent addition of nonmitogenic agents that, alone, have minimal effects on transcription in the absence of prior preconditioning by mitogen stimulation. These findings suggest that p300 is likely to play an important role in biological processes in which transcriptional bookmarking or preconditioning influences cellular growth and development through the dynamic priming of genes for response to rechallenge by secondary stimuli.gene regulation ͉ histone acetylation ͉ transcription ͉ ELL ͉ epigenetics

Five new isoprenylated flavones, artochamins A-E (1-5), together with eight known flavones (6-13), were isolated from the roots of Artocarpus chama. All structures were elucidated by spectroscopic methods. Artonin E (12) showed strong cytotoxicity against 1A9 (ovarian), significant activity against MCF-7 (breast adenocarcinoma), and moderate activity against HCT-8 (ileocecal) and MDA-MB-231 (breast adenocarcinoma) tumor cell lines. Artochamin C (3) was more potent against MCF-7, 1A9, HCT-8, and SK-MEL-2 (melanoma) than A549 (lung carcinoma), KB (epidermoid carcinoma of the nasopharynx), and its drug-resistant (KB-VIN) variant. Artocarpin (6) displayed weak but relatively broad inhibitory effects compared with 3 and 12.

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