Sharon Illenye

Cooperation of E2F-p130 and Sp1-pRb Complexes in Repression of the Chinese Hamster dhfr Gene

ABSTRACT In mammalian cells reiterated binding sites for Sp1 and two overlapping and inverted E2F sites at the transcription start site regulate the dhfr promoter during the cell growth cycle. Here we have examined the contributions of the dhfr Sp1 and E2F sites in the repression of dhfr gene expression. In serum-starved cells or during serum stimulation, the Chinese hamsterdhfr gene was not derepressed by trichostatin A (TSA), an inhibitor of histone deacetylases (HDAC). Immunoprecipitation experiments showed that HDAC1 and hypophosphorylated retinoblastoma protein (pRb) are associated with Sp1 in serum-starved CHOC400 cells. In transfection experiments, reporter plasmids containing the reiterated dhfr Sp1 sites were stimulated 10-fold by TSA, while a promoter containing four dhfr E2F sites and a TATA box was responsive to E2F but was completely unaffected by TSA. HDAC1 did not coprecipitate with p130-E2F DNA binding complexes, the predominant E2F binding activity in cell extracts after serum starvation, suggesting that p130 imposes a TSA-insensitive state on thedhfr promoter. In support of this notion, recruitment of GAL4-p130 to a dihydrofolate reductase-GAL4 reporter rendered the promoter insensitive to TSA, while repression by GAL4-pRb was sensitive to TSA. Upon phosphorylation of pRb and p130 after serum stimulation, the Sp1-pRb and p130-E2F interactions were lost while the Sp1-HDAC1 interaction persisted into S phase. Together these studies suggest a dynamic model for the cooperation of pRb and p130 in repression ofdhfr gene expression during withdrawal from the cell cycle. We propose that, during initial phases of cell cycle withdrawal, the binding of dephosphorylated pRb to Sp1-HDAC1 complexes and complexes of E2F-1 -to -3 with DP results in transient, HDAC-dependent suppression of dhfr transcription. Upon withdrawal of cells into G0, recruitment of p130 to E2F-4–DP-1 complexes at the transcription start site results in a TSA-insensitive complex that cooperates with Sp1-HDAC-pRb complexes to stably repressdhfr promoter activity in quiescent cells.

Protein-DNA interactions at the major and minor promoters of the divergently transcribed dhfr and rep3 genes during the Chinese hamster ovary cell cycle.

In mammals, two TATA-less bidirectional promoters regulate expression of the divergently transcribed dihydrofolate reductase (dhfr) and rep3 genes. In CHOC 400 cells, dhfr mRNA levels increase about fourfold during the G1-to-S phase transition of the cell cycle, whereas the levels of rep3 transcripts vary less than twofold during this time. To assess the role of DNA-binding proteins in transcriptional regulation of the dhfr and rep3 genes, the major and minor dhfr-rep3 promoter regions were analyzed by high-resolution genomic footprinting during the cell cycle. At the major dhfr promoter, prominent DNase I footprints over four upstream Sp1 binding sites did not vary throughout G1 and entry into the S phase. Genomic footprinting revealed that a protein is constitutively bound to the overlapping E2F sites throughout the G1-to-S phase transition, an interaction that is most evident on the transcribed template strand. On the nontranscribed strand, multiple changes in the DNase I cleavage pattern are observed during transit through G1 and entry into the S phase. By using gel mobility shift assays and a series of sequence-specific probes, two different species of E2F were shown to interact with the dhfr promoter during the cell cycle. The DNA binding activity of one E2F species, which preferentially recognizes the sequence TTTGGCGC, did not vary significantly during the cell cycle. The DNA binding activity of the second E2F species, which preferentially recognizes the sequence TTTCGCGC, increased during the G1-to-S phase transition. Together, these results indicate that Sp1 and the species of E2F that binds TTTGGCGC participate in the formation of a basal transcription complex, while the species of E2F that binds TTTCGCGC regulates dhfr gene expression during the G1-to-S phase transition. At the minor promoter, DNase I footprints at a consensus c-Myc binding site and three Sp1 binding sites showed little variation during the G1-to-S phase transition. In addition to protein binding at sequences known to be involved in the regulation of transcription, genomic footprinting of the entire promoter region also showed that a protein factor is constitutively bound to the first intron of the rep3 gene.

Accumulation of E2F-4.DP-1 DNA binding complexes correlates with induction of dhfr gene expression during the G1 to S phase transition.

Previously genomic DNase I footprinting showed changes in protein binding to two overlapping E2F sites correlates with activation of dhfr gene expression at the G1/S boundary of the Chinese hamster cell cycle (Wells, J., Held, P., Illenye, S., and Heintz, N. H. (1996) Mol. Cell. Biol. 16, 634-647). Here gel mobility and antibody supershift assays were used to relate changes in the components of E2F DNA binding complexes in cell extracts to repression and induction of dhfr gene expression. In extracts from log phase cells, E2F complexes contained predominantly E2F-4 and E2F-2 in association with DP-1, and DNA binding assays showed complexes containing E2F-2 preferentially interact with only one of the two overlapping E2F sites. In serum starvation-stimulation experiments, arrest in G1 by low serum was accompanied by decreased levels of dhfr mRNA and the appearance of an E2F-4·DP-1·p130 complex. After serum stimulation, induction of dhfr gene expression was preceded by loss of the p130 complex in mid G1 and coincided with marked increases in two free E2F·DP-1 complexes in late G1, one of which contained E2F-4 and a second which contained an unidentified E2F. We suggest activation of dhfr gene expression after serum stimulation requires at least two temporally distinct processes, relief of p130-mediated repression and subsequent activation of transcription by free E2F.

Transcriptional squelching by ectopic expression of E2F-1 and p53 is alleviated by proteasome inhibitors MG-132 and lactacystin

The transcription factors p53 and E2F-1 play important roles in the control of cell cycle progression. In transient transfection experiments, expression of E2F-1, other E2F family members, or p53 squelched transcription from cotransfected plasmids in a dose-dependent manner. Although the proteasome inhibitors MG-132 and lactacystin markedly increased the level of expression of E2F-1 and p53, these inhibitors completely alleviated squelching by both proteins. Several observations indicate MG-132 alleviates squelching by influencing the conformation of newly synthesized p53 and E2F-1: MG-132 increased the fraction of wild type p53 bound by a monoclonal antibody which preferentially recognizes mutant conformers of p53, increased binding of hsp70 to p53 and inhibited nuclear accumulation of both p53 and E2F-1, but not the pocket protein p107. The protease inhibitors ALLN and ALLM did not influence expression of E2F-1 or p53, nor did they alleviate squelching by either transcription factor. Because MG-132 and lactacycstin are highly specific inhibitors of the proteasome protease, our results suggest that the proteasome influences post-translational processes involved in proper folding and cytoplasmic clearing of E2F-1 and p53.

Functional analysis of bacterial artificial chromosomes in mammalian cells: Mouse Cdc6 is associated with the mitotic spindle apparatus

Bacterial artificial chromosomes (BACs) provide a well-characterized resource for studying the functional organization of genes and other large chromosomal domains. To facilitate functional studies in cell cultures, we have developed a simple approach for generating stable cell lines with variable copy numbers of any BAC. Here we describe hamster cell lines with BAC transgenes that express mouse Cdc6 at levels that correlate with BAC copy number; show that mouse Cdc6 is regulated normally during the cell cycle, binds chromatin, and is degraded during apoptosis; and report a novel fraction of Cdc6 that associates with the spindle apparatus during mitosis. With RNA interference to assess genetic complementation by BAC alleles, this system will facilitate functional studies on large chromosomal domains at variable copy number in mammalian cell models.

Association with E2F-1 governs intracellular trafficking and polyubiquitination of DP-1

The cell cycle-regulated transcription factor E2F is a family of heterodimers composed of E2F and DP protein subunits. While DP proteins stabilize DNA binding of E2F proteins, and influence the entry of E2F-4 and E2F-5 into the nucleus, the role of DP proteins in E2F-dependent gene expression is not well understood. Using immunolocalization, immunoprecipitation, and cell fractionation experiments, here we show association with E2F subunits governs intracellular trafficking and ubiquitination of DP-1. In transient transfection experiments, DP-1 polypeptides that stably bound E2F-1 entered the nucleus. DP-1 proteins that failed to associate with E2F subunits accumulated in the cell cytoplasm as polyubiquitinated DP-1. A Chinese hamster cell line that conditionally expresses HA-DP-1 was used to examine the effect of DP-1 on cell cycle progression. In serum response experiments, moderate increases in HA-DP-1 led to a threefold increase in E2F DNA binding activity in vitro, a corresponding increase in dhfr gene expression during transition of G1, and higher rates of S phase entry. However, flow cytometry showed cells expressing very high levels of HA-DP-1 failed to enter the S phase. Inhibition of cell cycle progression by high levels of HA-DP-1 was associated with the accumulation of other ubiquitinated cellular proteins, including c-jun and the cyclin-dependent kinase inhibitor p21, indicating that degradation of ubiquitinated proteins is required for progression from G0 to S phase even in the presence of activated E2F. Under similar conditions, expression of E2F-1 reduced the levels of ubiquitinated cellular proteins and accelerated cell cycle progression. Our studies indicate association with E2F subunits prevents ubiquitin-dependent degradation of DP-1 in the cytoplasm by promoting nuclear entry of E2F/DP heterodimers.

PCI-24781 (abexinostat), a novel histone deacetylase inhibitor, induces reactive oxygen species-dependent apoptosis and is synergistic with bortezomib in neuroblastoma.

In this study, we investigated the cytotoxic effects of a broad-spectrum histone deacetylase (HDAC) inhibitor, PCI-24781, alone and in combination with the proteasome inhibitor bortezomib in neuroblastoma cell lines. The combination was shown to induce synergistic cytotoxity involving the formation of reactive oxygen species. The cleavage of caspase-3 and PARP, as determined by western blotting, indicated that cell death was primarily due to apoptosis. Xenograft mouse models indicated increased survival among animals treated with this combination. The Notch signaling pathway and MYCN gene expression were quantified by reverse transcription-polymerase chain reaction (PCR) in cells treated with PCI-24781 and bortezomib, alone and in combination. Notch pathway expression increased in response to an HDAC inhibitor. NFKB1 and MYCN were both significantly down regulated. Our results suggest that PCI-24781 and bortezomib are synergistic in neuroblastoma cell lines and may be a new therapeutic strategy for this disease.

Caspase-dependent apoptosis by ectopic expression of E2F-4

E2F is a family of transcription factors which regulates cell cycle and apoptosis of mammalian cells. E2F-1-3 localize in the nucleus, and preferentially bind pRb, while E2F-4 and 5 have no nuclear localization signal and preferentially bind p107/p130. E2F-6 suppresses the transcriptional activity of other E2F proteins. DP-1 and 2 are heterodimeric partners of each E2F protein. Using tetracycline-responsive promoters, here we compared the effects of ectopic expression of E2F-1, DP-1 and E2F-4 on cell cycle progression and apoptosis in Chinese hamster cell lines. We found that E2F-4, as well as DP-1 and E2F-1, induced growth arrest and caspase-dependent apoptosis. E2F-4 did not have a marked effect on cell cycle progression, while E2F-1 induced DNA synthesis of resting cells and DP-1 arrested cells in G1. Ectopic expression of E2F-4 did not activate E2F-dependent transcription. Our results suggest that expression of E2F-4 at elevated levels induces growth arrest and apoptosis of mammalian cells through a mechanism distinct from E2F-1 and DP-1.

Oral RKS262 reduces tumor burden in a neuroblastoma xenograft animal model and mediates cytotoxicity through SAPK/JNK and ROS activation in vitro.

Patients diagnosed with high-risk neuroblastoma (NB), an extracranial solid tumor in children, have metastases and low survival (30%) despite aggressive multi-modal therapy. Therefore new therapies are urgently needed. We show significant in vitro and in vivo antitumor efficacy of RKS262 in NB. RKS262 showed superior cytotoxicity (IC50 = 6-25μM) against six representative NB cell lines compared to its parent analog Nifurtimox (currently in phase 2). Pre-formulated RKS262 (150mg/kg/daily) pellets administered orally, suppressed tumor growth (60%, p = 0.021) in NB xenograft mice within 28 days. RKS262-treated SMSKCNR cells showed TUNEL-positive DNA nicks and activation of ROS, MAPKs (SAPK/JNK), caspase-3, and p53, along with suppression of the IGF-1R/PI3K/PKC pathway and the Bcl2 family of proteins. RKS262 caused G2/M-phase arrest and suppressed cdc-2, cyclin B1, p21, and cyclin D1/D4 expression. N-acetyl-cysteine (NAC; 10mM) pre-treatment rescued cell viability of RKS262 (23µM)-treated SMSKCNR cells, and pre-treatment with ascorbic acid (100μM) and a MAPK inhibitor SB203580 (20μM) reversed SAPK/JNK, caspase-3 activation, PARP-1 cleavage, and suppression of IGF-1R, PI3K, and PKC phosphorylation. Further, treatment with exogenous BDNF (50nM) did not suppress SAPK/JNK or ROS activation due to RKS262. Rather, BDNF (50nM), EGF (100nM) and IGF-1 (100nM) co-treatment with RKS262 induced a remarkable S-phase arrest rather than a G2/M phase arrest when RKS262 was used alone. In summary, RKS262 shows oral efficacy in NB xenograft animals, and induces apoptosis in vitro in SMSKCNR cells via cell cycle arrest, MAPK and ROS activation, and suppression of IGF-1R/PI3K/PKC and Bcl2 family proteins in a growth factor (BDNF/EGF/IGF-1)-independent fashion.

Antitumor activity of nifurtimox is enhanced with tetrathiomolybdate in medulloblastoma.

Medulloblastoma, a neuroectodermal tumor arising in the cerebellum, is the most common brain tumor found in children. We recently showed that nifurtimox induces production of reactive oxygen species (ROS) and subsequent apoptosis in neuroblastoma cells both in vitro and in vivo. Tetrathiomolybdate (TM) has been shown to decrease cell proliferation by inhibition of superoxide dismutase-1 (SOD1). Since both nifurtimox and TM increase ROS levels in cells, we investigated whether the combination of nifurtimox and TM would act synergistically in medulloblastoma cell lines (D283, DAOY). Genome-wide transcriptional analysis, by hybridizing RNA isolated from nifurtimox and TM alone or in combination treated and control cells (D283) on Affymetrix exon array gene chips was carried out to further confirm synergy. We show that nifurtimox and TM alone and in combination decreased cell viability and increased ROS levels synergistically. Examination of cell morphology following drug treatment (nifurtimox + TM) and detection of caspase-3 activation via Western blotting indicated that cell death was primarily due to apoptosis. Microarray data from cells treated with nifurtimox and TM validated the induction of oxidative stress, as many Nrf2 target genes (HMOX1, GCLM, SLC7A11 and SRXN1) (p<10(-5)) were upregulated. Other genes related to apoptosis, oxidative stress, DNA damage, protein folding and nucleosome formation were differentially involved in cells following treatment with nifurtimox + TM. Taken together, our results suggest nifurtimox and TM act synergistically in medulloblastoma cells in vitro, and that this combination warrants further studies as a new treatment for medulloblastoma.