MiR-15 and miR-16 are direct transcriptional targets of E2F1 that limit E2F-induced proliferation by targeting cyclin E.

MicroRNAs (miR) are small noncoding RNA molecules that have recently emerged as critical regulators of gene expression and are often deregulated in cancer. In particular, miRs encoded by the miR-15a, miR-16-1 cluster seem to act as tumor suppressors. Here, we evidence that the miR-15a, miR-16-1 cluster and related miR-15b, miR-16-2 cluster comprise miRs regulated by E2F1, a pivotal transcription factor that can induce both proliferation and cell death. E2F1 is a critical downstream target of the tumor suppressor retinoblastoma (RB). The RB pathway is often inactivated in human tumors resulting in deregulated E2F activity. We show that expression levels of the 4 mature miRs, miR-15a, miR-16-1 and miR-15b, miR-16-2, as well as their precursor pri-miRNAs, are elevated upon activation of ectopic E2F1. Moreover, activation of endogenous E2Fs upregulates expression of these miRs and endogenous E2F1 binds their respective promoters. Importantly, we corroborate that miR-15a/b inhibits expression of cyclin E, the latter a key direct transcriptional target of E2F pivotal for the G(1)/S transition, raising the possibility that E2F1, miR-15, and cyclin E constitute a feed-forward loop that modulates E2F activity and cell-cycle progression. In support of this, ectopic expression of miR-15 inhibits the G(1)/S transition, and, conversely, inhibition of miR-15 expression enhances E2F1-induced upregulation of cyclin E1 levels. Furthermore, inhibition of both miR-15 and miR-16 enhances E2F1-induced G(1)/S transition. In summary, our data identify the miR-15 and miR-16 families as novel transcriptional targets of E2F, which, in turn, modulates E2F activity.

JNK activation is regulated by E2F and promotes E2F1-induced apoptosis.

Members of the E2F transcription factor family are critical downstream targets of the tumor suppressor RB and are often deregulated and hyperactive in human tumors. E2F regulates a diverse array of cellular functions including cell proliferation and apoptosis. Recent studies indicate that E2F also regulates expression of upstream components of pivotal signal transduction pathways, thereby modulating the activity of these pathways. We show here that E2F modulates the activity of the JNK pathway via E2F-induced upregulation of JNK phosphorylation. Accordingly, downregulating E2F1and E2F3 inhibits sustained UV-induced JNK phosphorylation and ectopic expression of E2F1 or E2F3 induces JNK phosphorylation and activation. The mechanism by which E2F modulates JNK phosphorylation involves transcriptional induction of the kinase GCK, a MAP4K that can activate JNK indirectly. Hence, inhibition of GCK expression impairs E2F1-induced JNK phosphorylation. The JNK pathway is an important mediator of stress-induced apoptosis and we show here that inhibition of JNK expression or activity significantly hinders E2F1-induced apoptosis. Overall, our data identify the kinase GCK as a novel E2F-regulated gene and reveal a functional link between a central signaling pathway, namely the JNK pathway, and the transcription factor E2F.

The tumor suppressor maspin mediates E2F1-induced sensitivity of cancer cells to chemotherapy.

The E2F1 transcription factor is a critical downstream target of the tumor suppressor RB. When activated, E2F1 can induce cell proliferation and/or apoptosis. In addition, E2F1 overexpression sensitizes cancer cells to chemotherapeutic drugs. In a screen for genes that are regulated synergistically by E2F1 and chemotherapy in cancer cells, we identified the proapoptotic tumor suppressor gene maspin (mammary serine protease inhibitor) as a novel E2F1-regulated gene. In line with being an E2F-regulated gene, maspin expression is inhibited by short hairpin RNA directed against E2F1 and increases upon activation of endogenous E2F. Furthermore, maspin mRNA and protein levels are elevated upon activation of exogenous E2F1. Importantly, we show that E2F1-mediated upregulation of maspin is enhanced by chemotherapeutic drugs, and inhibition of maspin expression significantly impairs the ability of E2F1 to promote chemotherapy-induced apoptosis. Summarily, our data indicate that maspin is an important effector of E2F1-induced chemosensitization.

Oxidative stress in Synechococcus sp. strain PCC 7942: various mechanisms for H2O2 detoxification with different physiological roles.

This study focuses on the mechanisms for hydrogen peroxide detoxification in Synechococcus sp. strain PCC 7942. To gain better understanding of the role of different routes of hydrogen peroxide detoxification, we inactivated TplA (thioredoxin-peroxidase-like), which we recently identified. In addition, we inactivated the gene encoding catalase-peroxidase and examined the ability to detoxify H(2)O(2) and to survive oxidative stress in both of the single mutants and in the double mutant. Surprisingly, we observed that the double mutant survived H(2)O(2) concentrations that the single catalase-peroxidase mutant could not tolerate. This phenotype correlated with an increased ability of the double mutant to detoxify externally added H(2)O(2) compared to the catalase-peroxidase mutant. Therefore, our studies suggested the existence of a hydrogen peroxide detoxification activity in addition to catalase-peroxidase and thioredoxin-peroxidase. The rate of detoxification of externally added H(2)O(2) was similar in the wild-type and the TplA mutant cells, suggesting that, under these conditions, catalase-peroxidase activity was essential for this process and TplA was dispensable. However, during excessive radiation, conditions under which the cell might experience oxidative stress, TplA appears to be essential for growth, and cells lacking it cannot compete with the wild-type strain. Overall, these studies suggested different physiological roles for various cellular hydrogen peroxide detoxification mechanisms in Synechococcus sp. strain PCC 7942.