Opposite DNA Strands Research Articles

Induction of Base Damages Representing a High Risk Site for Double-strand DNA Break Formation in Genomic DNA by Exposure of Cells to DNA Damaging Agents

DNA repair is known as a defense mechanism against genotoxic insults. However, the most lethal type of DNA damages, double-strand DNA breaks (DSBs), can be produced by DNA repair. We have previously demonstrated that when long patch base excision repair attempts to repair a synthetic substrate containing two uracils, the repair produces DSBs (Vispe, S. and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392 and Vispe, S., Ho, E. L., Yung, T. M., and Satoh, M. S. (2003) J. Biol. Chem. 278, 35279-35285). In this synthetic substrate, the two uracils are located on the opposite DNA strands (separated by an intervening sequence stable at 37 degrees C) and represent a high risk site for DSB formation. It is not clear, however, whether similar high risk sites are also induced in genomic DNA by exposure to DNA damaging agents. Thus, to investigate the mechanisms of DSB formation, we have modified the DSB formation assay developed previously and demonstrated that high risk sites for DSB formation are indeed generated in genomic DNA by exposure of cells to alkylating agents. In fact, genomic DNA containing alkylated base damages, which could represent high risk sites, are converted into DSBs by enzymes present in extracts prepared from cells derived from clinically normal individuals. Furthermore, DSBs are also produced by extracts from cells derived from ataxia-telangiectasia patients who show cancer proneness due to an impaired response to DSBs. These results suggest the presence of a novel link between base damage formation and DSBs and between long patch base excision repair and human diseases that occur due to an impaired response to DSB.

Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me 2+) ions, with the level of activity decreasing in the order Mn 2+ ≥ Mg 2+ > Ca 2+ ≥ Cu 2+ > Co 2+ ≥ Ni 2+ ≥ Zn 2+, whereas Fe 2+ did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me 2+ ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me 2+ ions in the 0.1–12 mM range, depending on the particular metal ion. In the presence of all Me 2+ ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu 2+, Zn 2+, or Ca 2+ inhibited the Mg 2+-dependent hydrolysis of scDNA, while Ni 2+, Co 2+, and Mn 2+ activated this reaction. The Mn 2+-dependent hydrolysis of scDNA was activated by Ca 2+, Ni 2+, Co 2+, and Mg 2+ ions but was inhibited by Cu 2+ and Zn 2+. After addition of the second metal ion, only in the case of Mg 2+ and Ca 2+ or Mn 2+ ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0–100%) in the presence of Mn 2+, Ca 2+, and Mg 2+ ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn 2+, Ca 2+, and Mg 2+ ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.

Rational design of an estrogen receptor mutant with altered DNA-binding specificity

Although artificial C2-H2 zinc fingers can be designed to recognize specific DNA sequences, it remains unclear to which extent nuclear receptor C4 zinc fingers can be tailored to bind novel DNA elements. Steroid receptors bind as dimers to palindromic response elements differing in the two central base pairs of repeated motifs. Predictions based on one amino acid—one base-pair relationships may not apply to estrogen receptors (ERs), which recognize the two central base pairs of estrogen response elements (EREs) via two charged amino acids, each contacting two bases on opposite DNA strands. Mutagenesis of these residues, E203 and K210 in ERα, indicated that both contribute to ERE binding. Removal of the electric charge and steric constraints associated with K210 was required for full loss of parental DNA-binding specificity and recognition of novel sequences by E203 mutants. Although some of the new binding profiles did not match predictions, the double mutation E203R-K210A generated as predicted a mutant ER that was transcriptionally active on palindromes of PuGCTCA motifs, but not on consensus EREs. This study demonstrates the feasibility of designing C4 zinc finger mutants with novel DNA-binding specificity, but also uncovers limitations of this approach.

Evx2-Hoxd13 Intergenic Region Restricts Enhancer Association to Hoxd13 Promoter

Expression of Hox genes is tightly regulated in spatial and temporal domains. Evx2 is located next to Hoxd13 within 8 kb on the opposite DNA strand. Early in development, the pattern of Hoxd13 expression resembles that of Evx2 in limb and genital buds. After 10 dpc, however, Evx2 begins to be expressed in CNS as well. We analyzed the region responsible for these differences using ES cell techniques, and found that the intergenic region between Evx2 and Hoxd13 behaves as a boundary element that functions differentially in space and time, specifically in the development of limbs, genital bud, and brain. This boundary element comprises a large sequence spanning several kilobases that can be divided into at least two units: a constitutive boundary element, which blocks transcription regulatory influences from the chromosomal environment, and a regulatory element, which controls the function of the constitutive boundary element in time and space.

The fibroblast growth factor-2 antisense gene inhibits nuclear accumulation of FGF-2 and delays cell cycle progression in C6 glioma cells

Fibroblast growth factor-2 (FGF-2) is a potent heparin-binding protein with growth-promoting and anti-apoptotic activity. Transcription of the GFG/NUDT6 gene on the opposite DNA strand generates an overlapping antisense RNA (FGF-AS) implicated in the post-transcriptional regulation of FGF-2. C6 glioma cells coordinately express FGF-2 and FGF-AS mRNA in a cell cycle-dependent manner. Cellular FGF-2 immunoreactivity was also cell cycle-dependent, with marked nuclear accumulation during S-phase. Stable transfection and overexpression of the FGF-AS RNA resulted in suppression of total cellular FGF-2, and a reduction in nuclear accumulation of FGF-2 isoforms. Serum stimulation of growth-arrested wild-type cells evoked a rapid nuclear translocation of FGF-2, and cell cycle re-entry. FGF-AS transfectants, in contrast, showed a significant delay in recovery of both nuclear FGF-2 staining and S-phase re-entry. Similar results were observed when cells were released from aphidicolin-induced G1 arrest or subjected to heat shock. These findings indicate that FGF-AS RNA inhibits expression and cell cycle-dependent nuclear accumulation of FGF-2, and this is associated with a marked delay in S-phase progression. The results suggest that the endogenous FGF antisense RNA may play a significant functional role in the regulation of FGF-2 dependent cell proliferation in FGF-2 expressing cells.

Microhomologies between T-DNA ends and target sites often occur in inverted orientation and may be responsible for the high frequency of T-DNA-associated inversions

Sequence analysis of left and right border integration sites of independent, single-copy T-DNA inserts in Arabidopsis thaliana revealed three previously unrecognized concomitants of T-DNA integration. First, genomic pre-insertion sites shared sequence similarity not only with the T-DNA left and right border regions, as was previously reported, but also at high frequency with the inverted complement of the T-DNA right border region. Second, palindromic sequences were frequently found to overlap or lie adjacent to genomic target sites, suggesting a high recombinogenic potential for palindromic elements during T-DNA integration and a possible role during the primary contact between the T-DNA and the target DNA. Third, "filler" DNA sequences between genomic pre-insertion site DNA and T-DNA often derive from sequences in the T-DNA left and right border regions that are clustered around palindromic sequences in these T-DNA regions, suggesting that these palindromic elements are "hot spots" for filler DNA formation. The discovery of inverted sequence similarities at the right border suggests a previously unrecognized mode of T-DNA integration that involves heteroduplex formation at both T-DNA borders and with opposite strands of the target DNA. Scanning for sequence similarities in both direct and inverted orientation may increase the probability and/or effectiveness of anchoring the T-DNA to the target DNA. Variations on this scheme may also account for inversion events at the target site of T-DNA integration and inverted T-DNA repeat formation, common sequence organization patterns associated with T-DNA integration.

Deletion of theparkinandPACRGgene promoter in early-onset parkinsonism

Autosomal recessive mutations in the parkin gene (PARK2) have been identified as a common cause of familial and also sporadic, early-onset parkinsonism (EOPD): point mutations, exonic deletions, and duplications or triplications have been described. Here we report a novel mutation, consisting of a deletion of the promoter and exon 1 of parkin (c.1-?_7+?del), in a family compatible with autosomal recessive EOPD and an isolated case. The former was compound heterozygous for the parkin c.1-?_7+?del mutation and an exon 3 deletion (c.172-?_412+?del). The latter was homozygous for the parkin c.1-?_7+?del mutation. The promoter region is shared by parkin and the neighboring parkin coregulated gene (PACRG), which are oriented head-to-head and are transcribed on opposite DNA strands. There were no parkin transcripts in lymphoblasts from the patients carrying the parkin c.1-?_7+?del mutation. The phenotypes of patients with promoter deletions and consequently absence of parkin and possibly PACRG expression, were similar to and no more severe than those of other EOPD patients with parkin mutations.

In vivo Expression of a Group I Intron HEG from the Antisense Strand of Didymium Ribosomal DNA

Two different isolates of the myxomycete Didymium iridis harbour homing endonuclease genes that are expressed from group I introns inserted into identical sites within the small subunit ribosomal DNA. The homing endonuclease proteins are related in sequence, and their gene structures share similar features such as the presence of small spliceosomal introns and functional polyadenylation sites. However, they are transcribed from opposite strands of the ribosomal DNA and presumable by different RNA polymerases. We have previously described the in vivo expression of the I-DirI homing endonuclease from within the ribosomal RNA precursor. In this paper, we demonstrate the in vivo expression of the I-DirII homing endonuclease from the opposite strand of the Didymium rRNA gene. A comparison of the expression strategies of the two genes demonstrates the feasibility of antisense expression and provides insight into nucleolar gene expression.

Potential regulatory SNPs in promoters of human genes: A systematic approach

Single nucleotide polymorphisms (SNPs) can significantly contribute to the cellular level of the mRNA transcripts encoded by human disease related genes. DNA variations between individuals can be an indication of predisposition to disease or affect the response to treatment. An algorithm allowing in silico extraction of SNPs with the high probability of influencing the level of gene expression is highly desirable. We performed a whole-genome analysis of SNP markers in regulatory areas of the human genes. Computational criteria were applied to predict an influence of the nucleotide replacement on the individual gene's expression. We formed a list of 14127 regulatory SNPs corresponding to 8555 regulatory areas suitable for future association studies. A catalogue of 1859 SNP entries, confirmed by analysis in populations, and allocated to 1607 human regulatory areas was created. We also revealed 13 cases of overlapped promoters corresponding to the human genes transcribed from opposite DNA strands and containing the regulatory SNP markers validated in populations. A population-validated set of regulatory SNP markers is organized in a database available in open access as a Supplementary file and by ftp://194.67.85.195/.

PAX2 Activates WNT4 Expression during Mammalian Kidney Development

The transcription factor PAX2 is expressed during normal kidney development and is thought to influence outgrowth and branching of the ureteric bud. Mice with homozygous null Pax2 mutations have developmental defects of the midbrain-hindbrain region, optic nerve, and ear and are anephric. During nephrogenesis, PAX2 is also expressed by mesenchymal cells as they cluster and reorganize to form proximal elements of each nephron, but the function of PAX2 in these cells is unknown. In this study we hypothesized that PAX2 activates expression of WNT4, a secreted glycoprotein known to be critical for successful nephrogenesis. PAX2 protein was identified in distal portions of the "S-shaped" body, and the protein persists in the emerging proximal tubules of murine fetal kidney. PAX2 activated WNT4 promoter activity 5-fold in co-transfection assays with JTC12 cells derived from the proximal tubule. Inspection of the 5'-flanking sequence of the human WNT4 gene identified three novel PAX2 recognition motifs; each exhibited specific PAX2 protein binding in electromobility shift assays. Two motifs were contained within a completely duplicated 0.66-kb cassette. Transfection of JTC12 cells with a PAX2 expression vector was associated with a 7-fold increase in endogenous WNT4 mRNA. In contrast, Wnt4 mRNA was decreased by 60% in mesenchymal cell condensates of fetal kidney from mice with a heterozygous Pax2 mutation. We speculated that a key function of PAX2 is to activate WNT4 gene expression in metanephric mesenchymal cells as they differentiate to form elements of the renal tubules.

BENA435, a new cell-permeant photoactivated green fluorescent DNA probe

N′-(2,8-Dimethoxy-12-methyl-dibenzo [c,h] [1,5] naphthyridin-6-yl)-N,N-dimethyl-propane-1,3-diamine (BENA435) is a new cell-membrane permeant DNA dye with absorption/emission maxima in complex with DNA at 435 and 484 nm. This new reagent is unrelated to known DNA dyes, and shows a distinct preference to bind double-stranded DNA over RNA. Hydrodynamic studies suggest that BENA435 intercalates between the opposite DNA strands. BENA435 fluoresces much stronger when bound to dA/dT rather than dG/dC homopolymers. We evaluated 14 related dibenzonaphthyridine derivatives and found BENA435 to be superior in its in vivo DNA-binding properties. Molecular modelling was used to develop a model of BENA435 intercalation between base pairs of a DNA helix. BENA435 fluorescence in the nuclei of cells increases upon illumination, suggesting photoactivation. BENA435 represents thus the first known cell-permeant photoactivated DNA-binding dye.

Spectrum of Complex DNA Damages Depends on the Incident Radiation

Ionizing radiation induces bistranded clustered damages--two or more abasic sites, oxidized bases and strand breaks on opposite DNA strands within a few helical turns. Since clusters are refractory to repair and are potential sources of double-strand breaks (DSBs), they are potentially lethal and mutagenic. Although induction of single-strand breaks (SSBs) and isolated lesions has been studied extensively, little is known about the factors affecting induction of clusters other than DSBs. To determine whether the type of incident radiation could affect the yields or spectra of specific clusters, we irradiated genomic T7 DNA, a simple 40-kbp linear, blunt-ended molecule, with ion beams [iron (970 MeV/nucleon), carbon (293 MeV/nucleon), titanium (980 MeV/nucleon), silicon (586 MeV/nucleon), protons (1 GeV/nucleon)] or 100 kVp X rays and then quantified DSBs, Fpg-oxypurine clusters and Nfo-abasic clusters using gel electrophoresis, electronic imaging and number average length analysis. The yields (damages/Mbp Gy(-1)) of all damages decreased with increasing linear energy transfer (LET) of the radiation. The relative frequencies of DSBs compared to abasic and oxybase clusters were higher for the charged particles-including the high-energy, low-LET protons-than for the ionizing photons.

Significance of 8-oxoG in the spectrum of DNA damages caused by ionising radiation of different quality

The yields and composition of DNA damages caused by ionising radiation depends on radiation quality. With increasing light energy transfer (LET), the proportion of isolated DNA damages with respect to cluster damaged sites decreases. Non-double strand break complex damages are induced by gamma radiation in mammalian cells at least four times more frequently that prompt DSB. The most important product of oxidative damage to DNA bases is 8-oxo-7,8-dihydroguanine (8-oxoG). The modelling of DNA damage induced by ionising radiation of different qualities was performed to assess frequencies and composition of complex damages containing 8-oxoG. The occurrence of clusters containing 8-oxoG increases from 6 to 11% for LET in the range 0.4-160 keV microm(-1). Distributions of single strand break (SSB) on opposite DNA strand around induced 8-oxoG have similar shape for different ionising radiations, but differ in their occurrence in the whole spectrum of DNA damages. The most probable configuration is a strand break localised at position +/-3 bases from 8-oxoG.

Increased stability and specificity through combined hybridization of peptide nucleic acid (PNA) and locked nucleic acid (LNA) to supercoiled plasmids for PNA-anchored “Bioplex” formation

Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as “openers” to partially overlapping sites on the opposite DNA strand. The PNA “opener” stabilized the binding of “linear” PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as “openers” to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA–peptides combined with shorter incubation times reduces unspecific binding and facilitates purification.

Structure and Function of the Type 3 Deiodinase Gene

Thyroid hormones (TH) are essential for normal growth and development in vertebrates, and are important for the maintenance of normal metabolic activity in most tissues of the body. Because the actions of TH result from the binding of 3,3',5'-triiodothyronine (T(3)) to specific nuclear receptors in the target cell, the extent of TH action in a given cell is dependent in part on the intracellular concentration of T(3). The type 3 deiodinase (D3) is a selenoenzyme that inactivates TH by catalyzing their conversion to biologically inactive metabolites. The findings that D3 activity is very high in the pregnant uterus and fetoplacental unit, and that D3-deficient mice exhibit deficits in growth, viability, and fertility strongly suggest that D3 plays an important role in development. The D3 gene (Dio3) is preferentially expressed from the paternally inherited allele and is associated with an overlapping gene transcribed from the opposite DNA strand (Dio3os). D3 mRNA expression and D3 activity are regulated by a number of hormones and growth factors as well as by genomic imprinting. Although some genomic structures appear to mediate some of these effects, many details concerning the function of the Dio3 gene are unresolved. These include the full characterization of the Dio3 and Dio3os genes, the elucidation of the mechanisms responsible for the developmental and tissue-specific patterns observed in Dio3 allelic expression, and the response of the genes to hormones and growth factors. Knowledge of these details will be important for understanding the physiologic function of an enzyme that appears to be critical for normal mammalian development.

Unusual Group II Introns in Bacteria of theBacillus cereusGroup

A combination of sequence and structure analysis and reverse transcriptase PCR experiments was used to characterize the group II introns in the complete genomes of two strains of the pathogen Bacillus cereus. While B. cereus ATCC 14579 harbors a single intron element in the chromosome, B. cereus ATCC 10987 contains three introns in the chromosome and four in its 208-kb pBc10987 plasmid. The most striking finding is the presence in B. cereus ATCC 10987 of an intron [B.c.I2(a)] located on the reverse strand of a gene encoding a putative cell surface protein which appears to be correlated to strains of clinical origin. Because of the opposite orientation of B.c.I2(a), the gene is disrupted. Even more striking is that B.c.I2(a) splices out of an RNA transcript corresponding to the opposite DNA strand. All other intragenic introns studied here are inserted in the same orientation as their host genes and splice out of the mRNA in vivo, setting the flanking exons in frame. Noticeably, B.c.I3 in B. cereus ATCC 10987 represents the first example of a group II intron entirely included within a conserved replication gene, namely, the alpha subunit of DNA polymerase III. Another striking finding is that the observed 3' splice site of B.c.I4 occurs 56 bp after the predicted end of the intron. This apparently unusual splicing mechanism may be related to structural irregularities in the 3' terminus. Finally, we also show that the intergenic introns of B. cereus ATCC 10987 are transcribed with their upstream genes and do splice in vivo.

Repair of Tandem Base Lesions in DNA by Human Cell Extracts Generates Persisting Single-strand Breaks

Clustered DNA damage, where two or more lesions are located proximal to each other on the same or opposite DNA strands, is frequently produced as a result of exposure to ionising radiation. It has been suggested that such complex damaged sites pose problems for repair pathways. In this study, we addressed the question of how two 8-oxoguanine lesions, located two nucleotides apart on the same DNA strand, are repaired. We find that in human cell extracts repair of either of the 8-oxoguanine lesions within a tandem damaged site is initiated randomly and that the majority of the initiated repair proceeds to completion. However, a fraction of the initiated repair is delayed at the stage of an incised AP site and the rate of further processing of this incised AP site is dependent on the position of the remaining 8-oxoguanine. If the remaining 8-oxoguanine residue is located near the 5' terminus of the incised abasic site, repair continues as efficiently as repair of a single 8-oxoguanine residue. However, repair is delayed after the incision step when the remaining 8-oxoguanine residue is located near the 3' terminus. Although the presence of the 8-oxoguanine residue near the 3' terminus did not affect either DNA polymerase beta activity or poly(ADP)ribose polymerase-1 affinity and turnover on an incised AP site, we find that 8-oxoguanine-DNA glycosylase has reduced ability to remove an 8-oxoguanine residue located near the 3' terminus of the incised AP site. We find that binding of the 8-oxoguanine-DNA glycosylase to this 8-oxoguanine residue inhibits DNA repair synthesis by DNA polymerase beta, thus delaying repair. We propose that interference between a DNA glycosylase and DNA polymerase during the repair of tandem lesions may lead to accumulation of the intermediate products that contain persisting DNA strand breaks.

Interaction of Checkpoint Proteins Hus1/Rad1/Rad9 with DNA Base Excision Repair Enzyme MutY Homolog in Fission Yeast, Schizosaccharomyces pombe

The DNA glycosylase MutY homolog (MYH) is responsible for removing adenines misincorporated opposite DNA strands containing guanine or 7,8-dihydro-8-oxoguanine by base excision repair thereby preventing G:C to T:A mutations. MYH has been shown to interact with the proliferating cell nuclear antigen (PCNA) in both human and fission yeast Schizosaccharomyces pombe systems. Here we show that S. pombe (Sp) MYH physically interacts with all subunits of the PCNA-like checkpoint protein heterotrimer, SpRad9/SpRad1/SpHus1, in yeast extracts and when the individual subunits are expressed in bacteria. The SpHus1 and SpPCNA binding sites are located in discrete regions of SpMYH. Immunoprecipitation assays reveal that the interaction between SpHus1 and SpMYH increases dramatically after hydrogen peroxide treatment, and this increase in the SpHus1-SpMYH interaction correlates with the presence of SpHus1 phosphorylation. In contrast, the interaction between SpPCNA and SpMYH after hydrogen peroxide treatment remains nearly unchanged. SpMYH associates with SpHus1 in a complex of approximately 450 kDa, the reported native molecular mass of the SpRad9/SpRad1/SpHus1-MYC complex. A larger portion of SpMYH shifts to the 150-500-kDa regions after hydrogen peroxide treatment in comparison with untreated extracts. SpHus1 phosphorylation is substantially reduced in SpMYH Delta cells after hydrogen peroxide treatment. These data suggest that MYH may act as an adaptor to recruit checkpoint proteins to the DNA lesions.

GENE impedance: a natural process for control of gene expression and the origin of RNA interference

Gene expression is controlled by coordinated transcriptional and post-transcriptional mechanisms. Normally, expression of a gene switches on and off in response to specific physiological signals that are triggered by cellular demand for the gene products at a given time. Based on our previous studies and the scientific literature, we hypothesize that when a gene promoter switches to transcriptional repression mode, transcription of the gene ceases, and a small amount of double-stranded RNA (dsRNA) is synthesized by the RNA polymerase switching to the opposite DNA strand at the termination region of the gene. These dsRNA structures, which result from normal transcriptional repression, can then be processed into short interfering RNAs (siRNAs) within the nucleus. These molecules subsequently direct specific cleavage of the cognate mRNAs and interfere with their translation through sequence complementarily. We further hypothesize that cellular defense mechanisms invoked by invading genetic elements could be rooted in this fundamental regulatory pathway that we call “GENE impedance”, or simply, GENEi. Here, we present a working model that illustrates how transcription-termination and transcription-arrest can contribute to the regulation of gene expression via GENEi. In our model RNAi is only one component of GENEi, which is a more generalized mechanism of gene regulation.

SJG-136 (NSC 694501), a novel rationally designed DNA minor groove interstrand cross-linking agent with potent and broad spectrum antitumor activity: part 2: efficacy evaluations.

Pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136 (NSC 694501) selectively cross-links guanine residues located on opposite strands of DNA, and exhibits potent in vitro cytotoxicity. In addition, SJG-136 is highly active in vivo in hollow fiber assays. In the current investigation, SJG-136 was evaluated for in vivo efficacy in 10 tumor models selected on the basis of sensitivity of cells grown in the hollow fiber and in vitro time course assays: LOX IMVI and UACC-62 (melanomas); OVCAR-3 and OVCAR-5 (ovarian carcinomas); MDA-MB-435 (breast carcinoma); SF-295 and C-6 (gliomas); LS-174T (colon carcinoma); HL-60 TB (promyelocytic leukemia); and NCI-H522 (lung carcinoma). SJG-136 was active against small (150 mg) and large (250-400 mg) xenografts with tumor mass reductions in all 10 models. In addition, significant growth delays occurred in nine models, cell kill in six models ranged between 1.9 and 7.2 logs, and there were 1 to 4/6 tumor-free responses in six models. SJG-136 is active following i.v. bolus injections, as well as by 5-day continuous infusions. Of all of the schedules tested, bolus administrations for 5 consecutive days (qd x 5) conferred the greatest efficacy. SJG-136 is active over a wide dosage range in athymic mouse xenografts: on a qd x 5 schedule, the maximum-tolerated dose was approximately 120 microg/kg/dose (total dose: 0.6 mg/kg = 1.8 mg/m2) and the minimum effective dose in the most sensitive model (SF-295) was approximately 16 microg/kg/dose (total dose: 0.08 mg/kg = 0.24 mg/m2). Results of this study extend the initial in vivo observations reported in the reference above and confirm the importance of expediting more detailed preclinical evaluations on this novel agent in support of phase I clinical trials in the United Kingdom and the United States, which are planned to commence shortly.