Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells.

Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST-Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST-Z2 is able to condense 130-150 kb bacterial artificial chromosomes (BACs) into protein-DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein-BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR(-) cells by GST-Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST-Z2-mediated gene transfer. Because DNA condensation by GST-Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.

Cooperation of E2F-p130 and Sp1-pRb complexes in repression of the Chinese hamster dhfr gene.

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 hamster dhfr 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 the dhfr 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 of dhfr 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 G(0), 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 repress dhfr promoter activity in quiescent cells.

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.

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.

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.

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.

Nuclear localization of DP and E2F transcription factors by heterodimeric partners and retinoblastoma protein family members.

E2F is a family of transcription factors implicated in the regulation of genes required for progression through G1 and entry into the S phase. The transcriptionally active forms of E2F are heterodimers composed of one polypeptide encoded by the E2F gene family and one polypeptide encoded by the DP gene family. The transcriptional activity of E2F/DP heterodimers is influenced by association with the members of the retinoblastoma tumor suppressor protein family (pRb, p107, and p130). Here the intracellular distribution of E2F and DP proteins was investigated in transiently transfected Chinese hamster and human cells. In transfected cells, DP-1 did not accumulate in the nucleus unless it was coexpressed with the heterodimeric partners E2F-1, E2F-2, or E2F-3. Domain mapping experiments showed that regions of E2F-1 and DP-1 that are required for stable association of the two proteins were also required for nuclear localization of DP-1. Unlike E2F-1, -2, and -3, E2F-4 did not accumulate in the nucleus unless it was coexpressed with DP-2, p107 and p130, but not pRb, stimulated nuclear localization of E2F-4, either alone or in combination with DP-2. These results indicate that DP proteins preferentially associate with specific E2F partners, and suggest that the ability of specific E2F/DP heterodimers to localize in the nucleus contributes to the regulation of E2F activity.

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.

Homodinuclear (Pt,Pt) and heterodinuclear (Ru,Pt) metal compounds as DNA-protein cross-linking agents: potential suicide DNA lesions.

Homodinuclear (Pt,Pt) and heterodinuclear (Ru,Pt) metal compounds having the generalized formula M(a)NH2(CH)4NH2M(b) are shown to form specific DNA lesions which can efficiently cross-link proteins to DNA. In this study, the homodinuclear case is represented by M(a) = M(b) = [cis-Pt(Cl2)-(NH3)] and the heterodinuclear case is represented by M(a) = [cis-RuCl2(DMSO)3] and M(b) = [cis-PtCl2(NH3)]. Native and denaturing polyacrylamide gel electrophoresis was used to show the formation of ternary coordination complexes between the metal-treated 49-bp DNA fragment and the Escherichia coli UvrA and UvrB DNA repair proteins. Treatment with proteinase K results in loss of the DNA-protein cross-links. DNA-protein cross-links formed between UvrA and DNA previously modified with the dinuclear metal compounds are reversible with the reducing agent beta-mercaptoethanol. The DNA lesion responsible for efficient DNA-protein cross-linking is most probably a DNA-DNA interstrand cross-link in which each metal atom is coordinated with one strand of the DNA helix. The formation of DNA repair protein associated DNA cross-links, potential "suicide adducts", suggests a novel action mechanism for these anticancer compounds. In addition, these dinuclear metal compounds should be very useful agents for the investigation of a wide range of protein-DNA interactions.

Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay.

The polymerase chain reaction (PCR) represents an alternative to the current methods for investigating DNA damage and repair in specific genomic segments. In theory, any DNA lesion which blocks Taq polymerase can be measured by this assay. We used quantitative PCR (QPCR) to determine the lesion frequencies produced by cisplatin and ultraviolet light (UV) in a 2.3 kilobase (kb) segment of mitochondrial DNA and a 2.6 kb segment of the DHFR gene in mouse leukemia L1210 cells. The frequency of UV-induced lesions increased linearly with dose, and was 0.58 lesions/10 kb/10 J/m2 in the mitochondrial DNA, and 0.37 lesions/10 kb/10 J/m2 in the DHFR gene. With cisplatin, the lesion frequency also increased linearly with dose, and was 0.17 lesions/10 kb/10 microM in the DHFR gene, and 0.07 lesions/10 kb/10 microM in mitochondrial DNA. This result is contrary to that of Murata et al., 1990 (1), in which mitochondrial DNA received greater cisplatin damage than did nuclear DNA. Using PCR to measure the repair of UV-induced lesions in the DHFR gene segment, we observed that less than 10% of the lesions were removed by 4 h, but over 70% of the lesions were removed by 8 h. Repair of 43% of UV-induced lesions in mitochondrial DNA was also observed during a 24 h period.

Induction of DNA strand breaks in cultured rat embryo cells by crocidolite asbestos as assessed by nick translation.

Asbestos, a proven carcinogen, is reported to have no genotoxic effects. We hypothesized, however, in light of its clastogenic effects that one mechanism by which asbestos induces cell transformation and tumorigenesis involves the induction of DNA strand scission. Cultured rat embryo cells were exposed to low concentrations of International Union Against Cancer crocidolite and examined at intervals ranging from 2 to 48 h. The induction of DNA strand breaks was examined using the technique of nick translation followed by autoradiography or scintillation counting. Our results indicate that cells exposed to crocidolite have a higher incidence of DNA breaks and that this effect becomes apparent within 2-6 hours of exposure. Ball-milled crocidolite as well as riebeckite have a significantly lower effect while glass fibers induce a more pronounced DNA strand damage. These observations support the role fiber length plays in carcinogenesis and suggest that the classification of asbestos as a nongenotoxic carcinogen be reconsidered.

Murine leukemia L1210 cell lines with different patterns of resistance to platinum coordination complexes.

A murine leukemia L1210 cell line has been developed that exhibits 30-fold resistance to 1,2-diaminocyclohexaneplatinum(II) analogs but retains sensitivity to cisplatin. This contrasts existing cell lines which exhibit the opposite pattern of resistance. Both cell lines retain their sensitivity to melphalan.