Search

ABT-263 inhibits the antiapoptotic proteins Bcl-2, Bcl-xL, and Bcl-w and has single-agent efficacy in numerous small cell lung carcinoma (SCLC) and leukemia/lymphoma cell lines in vitro and in vivo. It is currently in clinical trials for treating patients with SCLC and various leukemia/lymphomas. Identification of predictive markers for response will benefit the clinical development of ABT-263. We identified the expression of Bcl-2 family genes that correlated best with sensitivity to ABT-263 in a panel of 36 SCLC and 31 leukemia/lymphoma cell lines. In cells sensitive to ABT-263, expression of Bcl-2 and Noxa is elevated, whereas expression of Mcl-1 is higher in resistant cells. We also examined global expression differences to identify gene signature sets that correlated with sensitivity to ABT-263 to generate optimal signature sets predictive of sensitivity to ABT-263. Independent cell lines were used to verify the predictive power of the gene sets and to refine the optimal gene signatures. When comparing normal lung tissue and SCLC primary tumors, the expression pattern of these genes in the tumor tissue is most similar to sensitive SCLC lines, whereas normal tissue is most similar to resistant SCLC lines. Most of the genes identified using global expression patterns are related to the apoptotic pathway; however, all but Bcl-rambo are distinct from the Bcl-2 family. This study leverages global expression data to identify key gene expression patterns for sensitivity to ABT-263 in SCLC and leukemia/lymphoma and may provide guidance in the selection of patients in future clinical trials. Mol Cancer Ther; 9(3); 545–57

Aims Vitamin D deficiency is associated with cardiac hypertrophy and heart failure, and vitamin D therapy prevents the progression of cardiac hypertrophy in animal models. Here, we examine whether vitamin D therapy prevents progression of pre-existing cardiac hypertrophy and development of heart failure. Methods and results When male Dahl salt-sensitive rats were fed a high salt (HS) diet, all rats developed cardiac hypertrophy after 5 weeks. Thereafter, rats were treated with vehicle (V), paricalcitol (PC, an active vitamin D analogue, at 200 ng, IP 3x/week), enalapril (EP, 90 μg/day), and PC + EP. All groups were continued on the HS diet and evaluated after 4 weeks of therapy. The PC and PC + EP groups, but not the V and EP only groups, showed significant prevention of progression of pre-existing cardiac hypertrophy. The signs of decompensated heart failure were evident in the vehicle-treated group; these heart failure parameters significantly improved with PC, EP or PC + EP therapy. The expression of PKCα, which is regulated by Ca2+and known to stimulate cardiac hypertrophy, was significantly increased in the vehicle group, and PC, EP or PC + EP effectively decreased PKCα activation. We also observed normalization of genetic alterations during progression to heart failure with PC treatment. Conclusion PC treatment resulted in both the prevention of progression of pre-existing cardiac hypertrophy and the development of heart failure, compared with improvement in progression to heart failure by EP alone. These beneficial findings in heart were associated with inhibition of PKCα activation and reversal of gene alterations.

Both Akt and Aurora A kinase have been shown to be important targets for intervention for cancer therapy. We report here that Compound A (A-443654), a specific Akt inhibitor, interferes with mitotic progression and bipolar spindle formation. Compound A induces G2/M accumulation, defects in centrosome separation, and formation of either monopolar arrays or disorganized spindles. On the basis of gene expression array studies, we identified Aurora A as one of the genes regulated transcriptionally by Akt inhibitors including Compound A. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, either by PI3K inhibitor LY294002 or by Compound A, dramatically inhibits the promoter activity of Aurora A, whereas the mammalian target of rapamycin inhibitor has little effect, suggesting that Akt might be responsible for up-regulating Aurora A for mitotic progression. Further analysis of the Aurora A promoter region indicates that the Ets element but not the Sp1 element is required for Compound A-sensitive transcriptional control of Aurora A. Overexpression of Aurora A in cells treated with Compound A attenuates the mitotic arrest and the defects in bipolar spindle formation induced by Akt inhibition. Our studies suggest that that Akt may promote mitotic progression through the transcriptional regulation of Aurora A.

Observations in hemodialysis patients suggest a survival advantage associated with activated vitamin D therapy. Left ventricular (LV) structural and functional abnormalities are strongly linked with hemodialysis mortality. Here, we investigated whether paricalcitol (PC, 19-nor-1,25(OH) 2 D 2 ), an activated vitamin D compound, attenuates the development of LV abnormalities in the Dahl salt-sensitive (DSS) rat and whether humans demonstrate comparable findings. Compared with DSS rats fed a high-salt (HS) diet (6% NaCl for 6 weeks), HS+PC was associated with lower heart and lung weights, reduced LV mass, posterior wall thickness and end diastolic pressures, and increased fractional shortening. Blood pressures did not significantly differ between the HS groups. Plasma brain natriuretic peptide levels, and cardiac mRNA expression of brain natriuretic peptide, atrial natriuretic factor, and renin were significantly reduced in the HS+PC animals. Microarray analyses revealed 45 specific HS genes modified by PC. In a retrospective pilot study of hemodialysis patients, PC-treated subjects demonstrated improved diastolic function and a reduction in LV septal and posterior wall thickness by echocardiography compared with untreated patients. In summary, PC attenuates the development of LV alterations in DSS rats, and these effects should be examined in human clinical trials.

Activation of PKCtheta is associated with lipid-induced insulin resistance and PKCtheta knockout mice are protected from the lipid-induced defects. However, the exact mechanism by which PKCtheta contributes to insulin resistance is not known. To investigate whether an increase in PKCtheta expression leads to insulin resistance, C2C12 skeletal muscle cells were transfected with PKCtheta DNA and treated with different concentrations of insulin for 10 min. PKCtheta overexpression induced reduction of IRS-1 protein levels with a decrease in insulin-induced p85 binding to IRS-1, phosphorylation of PKB and its substrates, p70 and GSK3. Pretreatment of these cells with GF-109203X (a non-specific PKC inhibitor, IC50 for PKCtheta = 10 nM) recovered insulin signaling. PKCtheta was found to be expressed in liver and treatment of human hepatoma cells (HepG2) with high insulin and glucose resulted in an increase in PKCtheta expression that correlated with a decrease in IRS-1 protein levels and the development of insulin resistance. Reduction of PKCtheta expression using RNAi technology significantly inhibited the degradation of IRS-1 and enhanced insulin-induced IRS-1 tyrosine phosphorylation, p85 association to IRS-1 and PKB phosphorylation. In conclusion, by overexpressing PKCtheta or using RNAi technology to downregulate PKCtheta, we have demonstrated that PKCtheta has a key role in the development of insulin resistance. These findings suggest that PKCtheta mediates not only insulin resistance in muscle but also in liver, which may contribute to the development of whole body insulin resistance and diabetes.

Vitamin D analogs provide survival benefit for chronic kidney disease patients with cardiovascular complications. Activation of smooth muscle cells plays a role in cardiovascular diseases. It is not known how Vitamin D analogs modulate gene expression in smooth muscle cells. In this study, DNA microarray technology was used to assess the gene expression profile in human coronary artery smooth muscle cells treated with 0.1microM 1alpha,25-dihydroxyvitamin D3 (calcitriol) or paricalcitol (an analog of calcitriol) for 30 h. The effects of calcitriol and paricalcitol were similar. A total of 176 target genes were identified with 115 up-regulated and 61 down-regulated genes in the paricalcitol group. Target genes fall into various categories including cell differentiation/proliferation. Real-time RT-PCR analysis demonstrated that paricalcitol dose- and time-dependently regulated the expression of IGF1, WT1 and TGFbeta3, three genes known to modulate cell proliferation. Paricalcitol also down-regulated the expression of natriuretic peptide precursor B and thrombospondin 1. Both drugs inhibited cell proliferation in a dose-dependent manner. This study identified genes not previously known to be regulated by VDR, providing insight into understanding the role of VDR on regulating smooth muscle cell growth, thrombogenicity, fibrinolysis and endothelial regeneration.

Ras mutation has been detected in approximately 20-30% of all human carcinomas, primarily in pancreatic, colorectal, lung and bladder carcinomas. The indirect inhibition of Ras activity by inhibiting farnesyltransferase (FTase) function is one therapeutic intervention to control tumor growth. Here we report the preclinical anti-tumor activity of our most advanced FTase inhibitor (FTI), ABT-100, and a direct comparison with the current clinical candidates. ABT-100 is a highly selective, potent and orally bioavailable FTI. It broadly inhibits the growth of solid tumors in preclinical animal models. Thus, ABT-100 is an attractive candidate for further clinical evaluation. In addition, our results provide plausible insights to explain the impressive potency and selectivity of ABT-100. Finally, we have demonstrated that ABT-100 significantly suppresses the expression of vascular endothelial growth factor (VEGF) mRNA and secretion of VEGF protein, as well as inhibiting angiogenesis in the animal model.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its analogues have been shown to inhibit proliferation of human cancer cells mediated by vitamin D receptor (VDR). The over-expression of 25-hydroxyvitamin D-24-hydroxylase (CYP24A1), an enzyme involved in the metabolism of 1,25(OH)2D3 and its analogues, is associated with poor prognosis of some human cancers. In this study, we employed real-time reverse transcription PCR to examine the expression of VDR and CYP24A1 mRNA in a cohort of human breast, lung, colon and ovary tumor samples. We found that CYP24A1 mRNA was significantly up-regulated in colon, ovary and lung tumors, but down-regulated in breast tumor relative to the analogous normal tissues. As a comparison, VDR mRNA was modestly down-regulated in colon, breast and lung tumors, but highly up-regulated in ovarian tumors. Treatment of two breast cancer cell lines, SW-620 and MCF-7, and one colon cancer cell line, HT-29, by 1,25(OH)2D3 for 48 h profoundly stimulated CYP24A1 mRNA expression (EC50=0.6, 0.8 and 29.5 nM in SW-620, HT-29 and MCF-7, respectively), but did not significantly affect VDR mRNA expression. Growth as assessed by DNA synthesis was modestly arrested by 1,25(OH)2D3 after 72 h of incubation, but was not altered after a 5-day incubation period. These data suggest that the VDR signaling pathway may be compromised via the modulation of CYP24A1 and VDR in human tumors.

A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three 'top hits' from our screen have been demonstrated to result from off-target gene silencing. Two of the three 'siRNA hits' were found to directly trigger down-regulation of hif-1alpha mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1alpha mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1alpha was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3'-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving 'hits-follow-up' strategies for future large-scale knockdown experiments.

Short interfering RNA (siRNA) is widely used for studying gene function and holds great promise as a tool for validating drug targets and treating disease. A critical assumption in these applications is that the effect of siRNA on cells is specific, i.e., limited to the specific knockdown of the target gene. In this article, we characterize the specificity of siRNA by applying gene expression profiling. Several siRNAs were designed against different regions of the same target gene for three different targets. Their effects on cells were compared by using DNA microarrays to generate gene expression signatures. When the siRNA design and transfection conditions were optimized, the signatures for different siRNAs against the same target were shown to correlate very closely, whereas the signatures for different genes revealed no correlation. These results indicate that siRNA is a highly specific tool for targeted gene knockdown, establishing siRNA-mediated gene silencing as a reliable approach for large-scale screening of gene function and drug target validation.

Protein tyrosine phosphatase 1B (PTP1B) has been implicated as a negative regulator of insulin action. Overexpression of PTP1B protein has been observed in insulin-resistant states associated with obesity. Mice lacking a functional PTP1B gene exhibit increased insulin sensitivity and are resistant to weight gain. To investigate the role of PTP1B in adipose tissue from obese animals, hyperglycemic obese (ob/ob) mice were treated with PTP1B antisense oligonucleotide (ISIS-113715). A significant reduction in adiposity correlated with a decrease of PTP1B protein levels in fat. Antisense treatment also influenced the triglyceride content in adipocytes, correlating with a downregulation of genes encoding proteins involved in lipogenesis, such as sterol regulatory element-binding protein 1 and their downstream targets spot14 and fatty acid synthase, as well as other adipogenic genes, lipoprotein lipase, and peroxisome proliferator-activated receptor γ. In addition, an increase in insulin receptor substrate-2 protein and a differential regulation of the phosphatidylinositol 3-kinase regulatory subunit (p85α) isoforms expression were found in fat from antisense-treated animals, although increased insulin sensitivity measured by protein kinase B phosphorylation was not observed. These results demonstrate that PTP1B antisense treatment can modulate fat storage and lipogenesis in adipose tissue and might implicate PTP1B in the enlargement of adipocyte energy stores and development of obesity.

The Saccharomyces cerevisiae protein ELO2p is involved in the elongation of saturated and monounsaturated fatty acids. Among several sequences with limited identity with the S. cerevisiae ELO2 gene, a consensus cDNA sequence was identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. Human liver cDNA was amplified by PCR using oligonucleotides complementary to the 5' and 3' ends of the putative human cDNA sequence. The resulting full-length sequence, termed HELO1, consisted of 897 bp, which encoded 299 amino acids. However, in contrast with the ELO2 gene, expression of this open reading frame in S. cerevisiae demonstrated that the encoded protein was involved in the elongation of long-chain polyunsaturated fatty acids, as determined by the conversion of gamma-linolenic acid (C(18:3, n-6)) into dihomo-gamma-linolenic acid (C(20:3, n-6)), arachidonic acid (C(20:4, n-6)) into adrenic acid (C(22:4, n-6)), stearidonic acid (C(18:4, n-3)) into eicosatetraenoic acid (C(20:4, n-3)), eicosapentaenoic acid (C(20:5, n-3)) into omega3-docosapentaenoic acid (C(22:5, n-3)) and alpha-linolenic acid (C(18:3, n-3)) into omega3-eicosatrienoic acid (C(20:3, n-3)). The predicted amino acid sequence of the open reading frame had only 29% identity with the yeast ELO2 sequence, contained a single histidine-rich domain and had six transmembrane-spanning regions, as suggested by hydropathy analysis. The tissue expression profile revealed that the HELO1 gene is highly expressed in the adrenal gland and testis. Furthermore, the HELO1 gene is located on chromosome 6, best known for encoding the major histocompatibility complex, which is essential to the human immune response.

Two human expressed sequence tag (EST) cDNA sequences with identity with ∆ 5 - and ∆ 6 -desaturases from a filamentous fungus, Mortierella alpina , were identified from the LifeSeq ® database of Incyte Pharmaceuticals, Inc. (Palo Alto, CA, U.S.A.). An oligonucleotide complementary to the 3ʹ EST cDNA sequences was used to screen human liver cDNA using rapid amplification of cDNA ends (RACE)-PCR. The amplified DNA fragment had 98% identity with a putative open reading frame (ORF) predicted from a human genomic sequence, and encoded 444 amino acids. Expression of this ORF in mouse fibroblast cells demonstrated that the encoded protein was a ∆ 5 -desaturase, as determined by the conversion of dihomo-γ-linolenic acid (C 20:3, n -6 ) into arachidonic acid (C 20:4, n -6 ). The human ∆ 5 -desaturase contained a predicted N-terminal cytochrome b 5 -like domain, as well as three histidine-rich domains. A tissue expression profile revealed that this gene is highly expressed in fetal liver, fetal brain, adult brain and adrenal gland. A search of the existing databases led to localization of this ORF within a 14 kb interval flanked by the flap endonuclease-1 (FEN1) and vitelliform macular dystrophy (Best9s disease; VMD2) loci of chromosome 11q12.

Early detection of human immunodeficiency virus (HIV) in blood and blood products can be achieved by a sensitive nucleic acid amplification-based assay. We report on the performance of a PCR-based qualitative assay that detects both HIV type 1 (HIV-1) and HIV-2 with a sensitivity of 20 to 50 copies/ml. The assay has a specificity of 99.6% and an inhibition rate of 1.7%. One milliliter of sample is processed with a manifold system and Qiagen columns, and one-third of the extracted sample is used for PCR amplification. An internal control sequence, which is processed and amplified with each sample, monitors for amplification inhibition. Samples are reverse transcribed and are then amplified by reverse transcription-coupled PCR, after which HIV-1- and HIV-2-specific probes are hybridized to the amplified products. Following hybridization, samples are detected in the LCx instrument by microparticle enzyme immunoassay techniques. The detection system has an automated inactivation step that controls for PCR contamination. The HIV-1/2 qualitative RNA assay detects HIV-1 group M subtypes A, B, C, D, E, F, and G and group O. Testing of several HIV-1 seroconversion panels has demonstrated that the HIV-1/2 qualitative RNA assay detects HIV infection on the average of 6 days before p24 antigen can be detected and 11 days before antibodies can be detected.

We have characterized a stress-responsive transcriptional activation domain of mouse heat shock factor 1 (HSF1) by using chimeric GAL4-HSF1 fusion proteins. Fusion of the GAL4 DNA-binding domain to residues 124 to 503 of HSF1 results in a chimeric factor that binds DNA yet lacks any transcriptional activity. Transactivation is acquired upon exposure to heat shock or by deletion of a negative regulatory domain including part of the DNA-binding-domain-proximal leucine zippers. Analysis of a collection of GAL4-HSF1 deletion mutants revealed the minimal region for the constitutive transcriptional activator to map within the extreme carboxyl-terminal 108 amino acids, corresponding to a region rich in acidic and hydrophobic residues. Loss of residues 395 to 425 or 451 to 503, which are located at either end of this activation domain, severely diminished activity, indicating that the entire domain is required for transactivation. The minimal activation domain of HSF1 also confers enhanced transcriptional response to heat shock or cadmium treatment. These results demonstrate that the transcriptional activation domain of HSF1 is negatively regulated and that the signal for stress induction is mediated by interactions between the amino-terminal negative regulator and the carboxyl-terminal transcriptional activation domain.

To assess systematically the structural and functional aspects of histone gene transcription within a chromosomal context, we stably integrated an extensive set of human histone H4 gene constructs into mouse C127 cells. Levels of expression were determined by S1 nuclease protection assays for multiple mouse monoclonal cell lines containing these human H4 genes. For each cell line, we quantitated the number of integrated human H4 genes by Southern blot analysis. The results indicate that the expression of the human H4 gene is in part copy number dependent at low gene dosages. However, the level of expression varies among different cell lines containing similar numbers of copies of the same H4 gene construct. This result suggests that position-dependent chromosomal integration effects contribute to H4 gene transcription, consistent with the roles of long-range gene organization and nuclear architecture in gene regulation. At high copy number, the level of human H4 gene expression per copy decreased, and endogenous mouse H4 mRNA levels were also reduced. Furthermore, in vivo occupancy at the human H4 gene immediate 5' regulatory elements, as defined by genomic fingerprinting, showed copy number-dependent protein/DNA interactions. Hence, human and mouse H4 genes compete for titratable transcription factors in a cellular environment. Taken together, these results indicate cross-species compatibility and suggest limited representation in vivo of the factors involved in regulating histone H4 gene transcription.

Aβ in the form of soluble oligomers or amyloid deposits has been identified as an endogenous substance responsible for the induction of neurodegeneration in Alzheimer’s disease. To study Aβ-related processes, we evaluated both input-output electrophysiology and gene expression in hippocampi from Tg2576 and wild-type control mice 7 months of age. In hippocampal CA1 f-EPSP recordings, the postsynaptic response was reduced in Tg2576, while there was no significant difference in fiber volley amplitude or paired-pulse facilitation. Gene expression was evaluated on RNA extracted from hippocampi contralateral to those used for electrophysiology. Robust gene expression differences were observed between Tg2576 and WT hippocampi, many of which reflected deficits in glutamatergic synaptic transmission. These results support the identification of synaptic deficits in young adult Tg2576 mice, and identify gene products that potentially could be used as biomarkers for Aβ toxicity or as mechanistic targets in further studies

Multiple heat shock transcription factors (HSFs) have been discovered in several higher eukaryotes, raising questions about their respective functions in the cellular stress response. Previously, we had demonstrated that the two mouse HSFs (mHSF1 and mHSF2) interacted differently with the HSP70 heat shock element (HSE). To further address the issues of cooperativity and the interaction of multiple HSFs with the HSE, we selected new mHSF1 and mHSF2 DNA-binding sites through protein binding and PCR amplification. The selected sequences, isolated from a random population, were composed primarily of alternating inverted arrays of the pentameric consensus 5'-nGAAn-3', and the nucleotides flanking the core GAA motif were nonrandom. The average number of pentamers selected in each binding site was four to five for mHSF1 and two to three for mHSF2, suggesting differences in the potential for cooperative interactions between adjacent trimers. Our comparison of mHSF1 and mHSF2 binding to selected sequences further substantiated these differences in cooperativity as mHSF1, unlike mHSF2, was able to bind to extended HSE sequences, confirming previous observations on the HSP70 HSE. Certain selected sequences that exhibited preferential binding of mHSF1 or mHSF2 were mutagenized, and these studies demonstrated that the affinity of an HSE for a particular HSF and the extent of HSF interaction could be altered by single base substitutions. The domain of mHSF1 utilized for cooperative interactions was transferable, as chimeric mHSF1/mHSF2 proteins demonstrated that sequences within or adjacent to the mHSF1 DNA-binding domain were responsible. We have demonstrated that HSEs can have a greater affinity for a specific HSF and that in mice, mHSF1 utilizes a higher degree of cooperativity in DNA binding. This suggests two ways in which cells have developed to regulate the activity of closely related transcription factors: developing the ability to fully occupy the target binding site and alteration of the target site to favor interaction with a specific factor.

Previous in vivo studies have identified a prominent 4'-demethylepipodophyllotoxin-9-(4,6-O-thionylidine-beta-D-g lucopyranoside) (VM-26)-induced double-stranded topoisomerase II cleavage site at approximately +80 relative to the start of Drosophila hsp70 transcription (Kroeger, P. E., and Rowe, T. C. (1992) Biochemistry 31, 2492-2502). Topoisomerase II binding at this site correlated with the repression of hsp70 transcription suggesting that this protein-DNA interaction was important in the regulation of hsp70 gene expression. In this paper, we investigated the interaction of purified Drosophila topoisomerase II with a 271-base pair DNA fragment containing the +80 region of the hsp70 gene using the topoisomerase II-specific inhibitor VM-26. VM-26-induced topoisomerase II cleavage of the hsp70 DNA resulted in a major 4-base staggered double-stranded break at +84. In the absence of ATP the +84 site was the only significant VM-26-induced cleavage site. Addition of ATP to the reaction resulted in a stimulation of topoisomerase cleavage throughout the 271-base pair DNA fragment. Deletion analyses determined that approximately 15 to 25 bp of flanking sequence were required for efficient cleavage at most topoisomerase II sites within the hsp70 DNA. However, in the case of the +84 site, topoisomerase cleavage still occurred even when this site was split in half by the restriction enzyme PstI. Topoisomerase II cleavage of both "half-site" DNA molecules occurred at the correct positions on the 4-base single-stranded DNA overhangs generated by PstI. Cleavage was reversible indicating that topoisomerase II could reseal the single-stranded DNA break formed in each half-site substrate. Denaturation of the half-site molecules abolished topoisomerase II cleavage suggesting that cleavage required the duplex region adjacent to the single-stranded cleavage site. Identification of this unusual half-site substrate provides additional evidence that double-stranded cleavage of DNA by topoisomerase II occurs via two sequential single-stranded breaks.

To understand the function of multiple heat shock transcription factors in higher eukaryotes, we have characterized the interaction of recombinant mouse heat shock transcription factors 1 and 2 (mHSF1 and mHSF2) with their binding site, the heat shock element (HSE). For our analysis, we utilized the human HSP70 HSE, which consists of three perfect 5'-nGAAn-3' sites (1, 3, and 4) and two imperfect sites (2 and 5) arranged as tandem inverted repeats. Recombinant mHSF1 and mHSF2, which exist as trimers in solution, both bound specifically to this HSE and stimulated transcription of a human HSP70-CAT construct in vitro. Footprinting analyses revealed differential binding of mHSF1 and mHSF2 to the HSP70 HSE. Specifically, mHSF1 bound all five pentameric sites, whereas mHSF2 failed to interact with the first site of the HSE but bound to sites 2 to 5. Missing-nucleoside analysis demonstrated that the third and fourth nGAAn sites were essential for mHSF1 and mHSF2 binding. The binding of the initial mHSF1 trimer to the HSE exhibited preference for sites 3, 4, and 5, and then binding of a second trimer occurred at sites 1 and 2. These results suggest that HSF may recognize its binding site through the dyad symmetry of sites 3 and 4 but requires an adjacent site for stable interaction. Our data demonstrate that mHSF1 and mHSF2 bind specifically to the HSE through major groove interactions. Methidiumpropyl-EDTA footprinting revealed structural differences in the first and third repeats of the HSE, suggesting that the DNA is distorted in this region. The possibility that the HSE region is naturally distorted may assist in understanding how a trimer of HSF can bind to what is essentially an inverted repeat binding site.

ABT-263 inhibits the antiapoptotic proteins Bcl-2, Bcl-xL, and Bcl-w and has single-agent efficacy in numerous small cell lung carcinoma (SCLC) and leukemia/lymphoma cell lines in vitro and in vivo. It is currently in clinical trials for treating patients with SCLC and various leukemia/lymphomas. Identification of predictive markers for response will benefit the clinical development of ABT-263. We identified the expression of Bcl-2 family genes that correlated best with sensitivity to ABT-263 in a panel of 36 SCLC and 31 leukemia/lymphoma cell lines. In cells sensitive to ABT-263, expression of Bcl-2 and Noxa is elevated, whereas expression of Mcl-1 is higher in resistant cells. We also examined global expression differences to identify gene signature sets that correlated with sensitivity to ABT-263 to generate optimal signature sets predictive of sensitivity to ABT-263. Independent cell lines were used to verify the predictive power of the gene sets and to refine the optimal gene signatures. When comparing normal lung tissue and SCLC primary tumors, the expression pattern of these genes in the tumor tissue is most similar to sensitive SCLC lines, whereas normal tissue is most similar to resistant SCLC lines. Most of the genes identified using global expression patterns are related to the apoptotic pathway; however, all but Bcl-rambo are distinct from the Bcl-2 family. This study leverages global expression data to identify key gene expression patterns for sensitivity to ABT-263 in SCLC and leukemia/lymphoma and may provide guidance in the selection of patients in future clinical trials. Mol Cancer Ther; 9(3); 545–57

We have compared topoisomerase I and II cleavage sites on the actin 5C and 57A genes and the hsp70 genes in Drosophila Kc cells using the inhibitors camptothecin (topoisomerase I specific) and VM-26 (topoisomerase II specific) to assess the role of these enzymes in transcriptional regulation. Topoisomerase I cleavage sites were localized to the transcribed regions of the actin 5C and hsp70 genes and were present only when these genes were active. The actin 57A gene, shown previously to be inactive in Kc cells, had no detectable topoisomerase I cleavage sites. In contrast to topoisomerase I, topoisomerase II cleavage sites could be detected on transcriptionally active and inactive actin and hsp70 DNA sequences. Topoisomerase II cleavage sites on the inactive hsp70 gene were primarily localized to the very 5' end of the transcribed region of the gene. However, upon heat-induced activation of hsp70 transcription, topoisomerase II cleavage rapidly shifted from the 5' to the 3' end of the gene. Then, during the shutdown of hsp70 expression, there was a gradual reappearance of topoisomerase II cleavage at the 5' end of the gene that temporally correlated with the repression of hsp70 transcription. There was a similar preferential association of topoisomerase II with the 5' ends of transcriptionally repressed actin 5C and 57A genes. These results demonstrate that there are marked differences in how topoisomerases I and II interact with transcriptionally active and inactive regions of chromatin. In addition, we have identified an unusual type of topoisomerase II binding site that is preferentially associated with the 5' ends of inactive hsp70 and actin genes, suggesting that this enzyme may facilitate changes in chromatin structure that are associated with repression of gene transcription.

Vitamin D receptor (VDR) activation therapy is associated with cardiovascular/survival benefit in chronic kidney disease (CKD), but the mechanism of action is not well understood. The aim of this study is to investigate how uremia affects gene expression in aorta and whether VDR activation modulates the uremic effect. DNA microarray technology was used to assess the gene expression profile in aorta prepared from SHAM and 5/6 nephrectomized (NX) rat, a model of Stage 4/5 CKD, treated with or without 0.17 μg/kg paricalcitol, a VDR activator. As expected, paricalcitol at 0.17 μg/kg after two weeks of treatment effectively suppressed serum parathyroid hormone (PTH); no significant difference was observed in ionized calcium or serum phosphorus. Uremia exhibited a significant effect on the gene expression pattern in the aorta, affecting 468 sequences ( ≥1.5-fold changes with p

Aβ in the form of soluble oligomers or amyloid deposits has been identified as an endogenous substance responsible for the induction of neuro degeneration in Alzheimer’s disease. To study Aβ-related processes, we evaluated both input-output electrophysiology and gene expression in hippocampi from Tg2576 and wild-type control mice 7 months of age. In hippocampal CA1 f-EPSP recordings, the postsynaptic response was reduced in Tg2576, while there was no significant difference in fiber volley amplitude or paired-pulse facilitation. Gene expression was evaluated on RNA extracted from hippocampi contralateral to those used for electrophysiology. Robust gene expression differences were observed between Tg2576 and WT hippocampi, many of which reflected deficits in glutamatergic synaptic transmission. These results support the identification of synaptic deficits in young adult Tg2576 mice, and identify gene products that potentially could be used as biomarkers for Aβ toxicity or as mechanistic targets in further studies.

Clinical observations show that an increase in serum inorganic phosphorus (Pi) is linked to higher cardiovascular (CV) mortality, while vitamin D receptor (VDR) agonist therapy is associated with survival benefit in stage 5 chronic kidney disease. Smooth muscle cells (SMCs) play an important role in CV pathophysiology, but the interaction between Pi and the VDR signaling pathway in SMCs is not known. Real-time RT-PCR studies revealed that elevated Pi (2.06 mM) modulated VDR-mediated regulation of a panel of genes including thrombomodulin and osteopontin in SMCs. DNA microarray results demonstrated that increasing Pi from 0.9 to 2.06 mM exerted a widespread modulating effect on VDR-mediated gene expression. A total of 325 target genes were affected by paricalcitol at 0.9 mM Pi, with 195 up- and 130 downregulated. The number of target genes affected by paricalcitol at 2.06 mM Pi decreased to 86, with 55 up- and 31 downregulated. VDR-mediated gene expression in As4.1 cells (a juxtaglomerular cell-like cell line derived from kidney tumors in SV40 T-antigen transgenic mice) and peroxisome proliferator-activated receptor (PPAR)γ-mediated gene expression in SMCs were also altered by elevated Pi, suggesting that the observation is not unique to VDR in SMCs. Mechanism analysis showed that elevated Pi had no significant effect on VDR or PPARγ protein level but altered the cytosolic vs. nuclear distribution of NF-κB or nuclear receptor corepressor 1 (NCoR1). Our results demonstrate for the first time that elevated Pi affects VDR-mediated gene expression in human coronary artery SMCs and the effect is not limited to VDR in SMCs.

Vitamin D analogs such as paricalcitol and calcitriol that activate the vitamin D receptor (VDR) provide survival benefit for Stage 5 chronic kidney disease (CKD) patients, possibly associated with a decrease in cardiovascular (CV)-related incidents. Phenotypic changes of smooth muscle cells play an important role in CV disease. The role of vitamin D analogs in modulating gene expression in smooth muscle cells is still not well understood. In this study, DNA microarray analysis of approximately 22,000 different human genes was used to characterize the VDR-mediated gene expression profile in human coronary artery smooth muscle cells (CASMC) at rest. Cells in serum free medium were treated with 0.1 microM calcitriol (1alpha,25-dihydroxyvitamin D(3)) or paricalcitol (19-nor-1alpha,25-(OH)(2)D(2)) for 30 h. A total of 181 target genes were identified, with 103 genes upregulated and 78 downregulated (>two fold changes in either drug treatment group with P < 0.01). No significant difference was observed between calcitriol and paricalcitol. Target genes fell into various categories with the top five in cellular process, cell communication, signal transduction, development, and morphogenesis. Twenty-two selected genes linked to the CV system were also impacted. Real-time RT-PCR and/or Western blotting analysis were employed to confirm the expression patterns of selected genes such as 25-hydroxyvitamin D-24-hydroxylase, Wilms' tumor gene 1, transforming growth factorbeta3, plasminogen activator inhibitor-1, thrombospondin-1 (THBS1), and thrombomodulin (TM). This study provides insight into understanding the role of VDR in regulating gene expression in resting smooth muscle cells.

Background: The vitamin D receptor (VDR) is involved in the regulation of renin expression and vitamin D analogs down-regulated renin mRNA expression in As4.1 cells. Methods: Microarray analysis was used to assess the VDR-mediated gene expression profile in As4.1 cells treated with paricalcitol, followed by real-time RT-PCR. Mechanistic analyses were done using siRNA, electrophoretic mobility shift assay (EMSA) and Western blotting. Results: Microarray analysis shows that 709 target genes were affected by paricalcitol with 286 up- and 423 downregulated. A number of major pathways were impacted including transcription factors. Real-time RT-PCR confirmed the microarray results. Treatment of the cells with siRNA against nuclear receptor co-repressor (NCOR1) eliminated VDR-mediated renin suppression. Using EMSA, paricalcitol treatment reduced the level of protein complex binding to the cyclic AMP-responsive element (CRE)-like domain in the renin distal enhancer region. VDR, CRE-binding protein (CREB1) and NCOR1 were identified in the complex binding to the CRE-like domain by Western blot in the paricalcitol-treated cells. Paricalcitol treatment resulted in an increase in the VDR level, but no significant change in the CREB1 and NCOR1 levels. Conclusion: These results suggest that VDR-mediated renin suppression likely acts through a transcriptional regulatory complex including CREB1, NCOR1 and VDR that binds to the CRE-like domain in the renin enhancer region.

Transient receptor potential channel type V (TRPV) 1 is a non-selective cation channel that can be activated by capsaicin, endogenous vanilloids, heat and protons. The human TRPV1 splice variant, TRPV1b, lacking exon 7, was cloned from human dorsal root ganglia (DRG) RNA. The expression profile and relative abundance of TRPV1b and TRPV1 in 35 different human tissues were determined by quantitative RT-PCR using isoform-specific probes. TRPV1b was most abundant in fetal brain, adult cerebellum and DRG. Functional studies using electrophysiological techniques showed that recombinant TRPV1b was not activated by capsaicin (1 microM), protons (pH 5.0) or heat (50 degrees C). However, recombinant TRPV1b did form multimeric complexes and was detected on the plasma membrane of cells, demonstrating that the lack of channel function was not due to defects in complex formation or cell surface expression. These results demonstrate that exon 7, which encodes the third ankyrin domain and 44 amino acids thereafter, is required for normal channel function of human TRPV1. Moreover, when co-expressed with TRPV1, TRPV1b formed complexes with TRPV1, and inhibited TRPV1 channel function in response to capsaicin, acidic pH, heat and endogenous vanilloids, dose-dependently. Taken together, these data support the hypothesis that TRPV1b is a naturally existing inhibitory modulator of TRPV1.

Blockade of presynaptic histamine H(3) receptors with potent and selective ligands improves cognitive function in rodents and there is significant interest in developing such drugs for long-term symptomatic treatment of CNS disorders such as attention deficit hyperactivity disorder (ADHD). Unfortunately, little is known about the effects of repeated exposure to H(3) receptor antagonists/inverse agonists. We therefore investigated the effects of acute and repeated daily administration of two potent, brain penetrating H(3) receptor antagonists/inverse agonists, ciproxifan and A-304121, on rat body weight, food and water intake, core temperature and locomotor activity, as well as H(3) receptor density and gene expression levels. Methylphenidate, used clinically for the treatment of ADHD, was included as an additional comparator. Ciproxifan, an imidazole-based compound, decreased food intake over the first 10 days and locomotor activity acutely, but these effects were lost after further repeated administration. The ex vivo binding studies revealed increased H(3) receptor density in rats following repeated administration of ciproxifan for 10 or 15 days; however, H(3) receptor gene expression was not changed. In contrast, rats treated with the non-imidazole, A-304121, did not differ from controls on any measure during the observation period, while rats treated with methylphenidate exhibited hyperthermia and hyperactivity. The implications for potential long-term treatment with H(3) receptor antagonists in CNS disorders such as ADHD are discussed.

Vitamin D3 is modified by vitamin D3-25-hydroxylase in the liver, and 25-hydroxyvitamin D3-1alpha-hydroxylase in the kidney, to form the active metabolite, 1,25-dihydroxyvitamin D3. Chronic kidney disease (CKD) is characterized by reduced synthesis of 1,25-dibydroxyvitamin D3, inadequate renal phosphate clearance and calcium imbalance, secondary hyperparathyroidism (SHPT) and bone disease. CKD patients encounter a much higher risk of cardiovascular disease (CVD) than the general public. The cardiovascular risk factors for CKD patients include conventional factors such as age, gender, hypertension, diabetes, dyslipidemia and smoking, and non-conventional factors, such as anemia, uremia, reduced vascular compliance, inflammation and various hormonal factors. Several vitamin D analogs are currently available for the treatment of SHPT, and recent clinical data show that these analogs provide survival benefit for CKD patients in the order of paricalcitolcalcitriolno vitamin D analog, independent of parathyroid hormone and calcium. Moreover, the survival benefit seems to be associated with cardiovascular causes. The observations made from these clinical studies raised intriguing questions about the involvement of the vitamin D receptor locus (VDR) in the cardiovascular system. This review discusses recent data regarding the role of vitamin D and its analogs in the CVD associated with CKD.

The profile of tetrodotoxin sensitive (TTX-S) and resistant (TTX-R) Na(+) channels and their contribution to action potentials and firing patterns were studied in isolated small dorsal root ganglion (DRG) neurons after L5/L6 spinal nerve ligation (SNL). Total TTX-R Na(+) currents and Na(v) 1.8 mRNA were reduced in injured L5 DRG neurons 14 days after SNL. In contrast, TTX-R Na(+)currents and Na(v) 1.8 mRNA were upregulated in uninjured L4 DRG neurons after SNL. Voltage-dependent inactivation of TTX-R Na(+) channels in these neurons was shifted to hyperpolarized potentials by 4 mV. Two types of neurons were identified in injured L5 DRG neurons after SNL. Type I neurons (57%) had significantly lower threshold but exhibited normal resting membrane potential (RMP) and action potential amplitude. Type II neurons (43%) had significantly smaller action potential amplitude but retained similar RMP and threshold to those from sham rats. None of the injured neurons could generate repetitive firing. In the presence of TTX, only 26% of injured neurons could generate action potentials that had smaller amplitude, higher threshold, and higher rheobase compared with sham rats. In contrast, action potentials and firing patterns in uninjured L4 DRG neurons after SNL, in the presence or absence of TTX, were not affected. These results suggest that TTX-R Na(+) channels play important roles in regulating action potentials and firing patterns in small DRG neurons and that downregulation in injured neurons and upregulation in uninjured neurons confer differential roles in shaping electrogenesis, and perhaps pain transmission, in these neurons.

The molecular properties of the sulfonylurea receptor 2 (SUR2) subunits of K(ATP) channels expressed in urinary bladder were assessed by polymerase chain reaction (PCR). This showed that SUR2B exon 17- mRNA (72%) was predominant over the SUR2B exon 17+ splice variant (28%). The pharmacological properties of both of these isoforms stably expressed in mouse Ltk(-)cells (L-cells) with K(IR) 6.2 were determined by measuring changes in membrane potential responses evoked by K(+) channel openers using bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)) fluorescence. The rank order potency of a variety of structurally distinct K(+) channel openers was found to be the same in both stable cell lines and compared well with guinea pig bladder cells. The potency of these compounds in the SUR2B exon 17- cells more closely resembled the potency measured in guinea pig bladder unlike the cell line containing the SUR2B exon 17+ subtype. Analysis of the displacement of [125I]A-312110 binding with the same K(+) channel openers to the SUR2B exon 17- cells showed excellent correlation to those measured in guinea pig bladder. This study supports the notion that K(ATP) channels containing SUR2B exon 17- represent a major splice variant expressed in urinary bladder smooth muscle.

The task of specific gene knockdown in vitro has been facilitated through the use of short interfering RNA (siRNA), which is now widely used for studying gene function, as well as for identifying and validating new drug targets. We explored the possibility of using siRNA for dissecting cellular pathways by siRNA-mediated gene silencing followed by gene expression profiling and systematic pathway analysis. We used siRNA to eliminate the Rb1 gene in human cells and determined the effects of Rb1 knockdown on the cell by using microarray-based gene expression profiling coupled with quantitative pathway analysis using the GenMapp and MappFinder software. Retinoblastoma protein is one of the key cell cycle regulators, which exerts its function through its interactions with E2F transcription factors. Rb1 knockdown affected G1/S and G2/M transitions of the cell cycle, DNA replication and repair, mitosis, and apoptosis, indicating that siRNA-mediated transient elimination of Rb1 mimics the control of cell cycle through Rb1 dissociation from E2F. Additionally, we observed significant effects on the processes of DNA damage response and epigenetic regulation of gene expression. Analysis of transcription factor binding sites was utilized to distinguish between putative direct targets and genes induced through other mechanisms. Our approach, which combines the use of siRNA-mediated gene silencing, mediated microarray screening and quantitative pathway analysis, can be used in functional genomics to elucidate the role of the target gene in intracellular pathways. The approach also holds significant promise for compound selection in drug discovery.

Protein tyrosine phosphatases are important regulators of insulin signal transduction. Our studies have shown that in insulin resistant and diabetic ob/ob and db/db mice, reducing the levels of protein tyrosine phosphatase 1B (PTP1B) protein by treatment with a PTP1B antisense oligonucleotide resulted in improved insulin sensitivity and normalized plasma glucose levels. The mechanism by which PTP1B inhibition improves insulin sensitivity is not fully understood. We have used microarray analysis to compare gene expression changes in adipose tissue, liver and muscle of PTP1B antisense-treated ob/ob mice. Our results show that treatment with PTP1B antisense resulted in the downregulation of genes involved in lipogenesis in both fat and liver, and a downregulation of genes involved in adipocyte differentiation in fat, suggesting that PTP1B antisense acts through a different mechanism than thiazolidinedione (TZD) treatment. In summary, microarray results suggest that reduction of PTP1B may alleviate hyperglycemia and enhance insulin sensitivity by a different mechanism than TZD treatment.

In a search for novel early T cell activation transcripts, we identified expressed sequence tags (ESTs) more abundantly expressed in normal human CD4+ T lymphocytes fully activated by a 5 h exposure to CD3 plus CD28 mAbs, compared to the same cells stimulated with either CD3 mAb or CD28 mAb alone. An EST was identified that hybridized with a 1.7 kb transcript expressed in activated T cells but was undetectable by Northern blot analysis in resting T cells or other normal tissues. The T cell transcript was maximally induced within 6 h and remained elevated for at least 47 h. Induction of the transcript was blocked by cyclosporin A, FK506, and dexamethasone but not by rapamycin. The transcript was polyadenylated but lacked an open reading. A BLAST search of the NCBI database revealed that the transcript shared identity with the recently reported human BIC proto-oncogene that encodes a noncoding mRNA (W. Tam, Gene 274 (2001) 157). Our data demonstrate that transcriptional activation of the BIC proto-oncogene is an early and sustained T cell activation event and suggest an important role for noncoding mRNA in T cell function.

Protein tyrosine phosphatase 1B (PTP1B) has recently been implicated in the regulation of body weight. A surprising phenotype of PTP1B-deficient mice is their resistance to diet-induced obesity. Since leptin is one of the primary hormones involved in the regulation of body weight and energy homeostasis, we investigated whether PTP1B affects leptin receptor (lepR) signaling directly. A mouse hypothalamic cell line, GT1-7, was established as a suitable cell model for the study of leptin signaling. Stimulation of GT1-7 cells by leptin caused tyrosine phosphorylation of endogenous STAT3 and activation of a STAT-dependent luciferase reporter gene. Over-expression of PTP1B in GT1-7 cells resulted in a dose-dependent decrease in endogenous JAK2 and STAT3 tyrosine phosphorylation compared with cells transfected with lepR alone. Consistent with inhibition of JAK-STAT signaling, PTP1B over-expression caused a dose-dependent decrease in leptin-induced, STAT-dependent luciferase reporter gene activation in GT1-7 cells. Furthermore, over-expression of PTP1B led to a decrease in mRNA accumulation of suppressor-of-cytokine-signalling-3 (SOCS3) and c-fos, genes that are acutely induced by leptin. Using gene microarray analysis, we confirmed that PTP1B reduces the level of gene expression of SOCS3 and showed that the expression level of other leptin-regulated genes was affected. Genes up-regulated by leptin were decreased in cells over-expressing PTP1B. Conversely, the expression of genes down-regulated by leptin was enhanced by PTP1B over-expression in GT1-7 cells. Our findings indicate that PTP1B is a negative regulator of leptin signaling and suggest that PTP1B inhibitors might be efficacious in the treatment of obesity by increasing leptin sensitivity.

The distribution of human sulfonylurea receptor-2 (SUR2)-containing K(ATP) channels was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR). mRNA for SUR2B was detected in a variety of tissues including brain, skeletal, cardiac and smooth muscle, whereas SUR2A message was restricted to cardiac and skeletal muscle. An additional splice variant of SUR2 that lacked exon 17 was also identified by RT-PCR in tissues expressing both SUR2A and SUR2B or SUR2B alone. Quantification of RNA for SUR2 exon 17+ and SUR2 exon 17- splice variants using real-time Taqman PCR indicated differential levels of expression in brain, kidney, skeletal muscle, heart and small intestine. Interestingly, the SUR2 exon 17+ variant is the major species expressed in all tissues examined in this study. Each of the SUR2 splice variants transiently expressed with the inward rectifier Kir 6.2 formed functional K(ATP) channels in HEK 293 cells as assessed either by changes in DiBAC(4)(3) fluorescence responses or glyburide-sensitive whole cell currents. Collectively, our findings demonstrate that various SUR2 splice variants have distinct expression patterns and can form functional K(ATP) channels.

A model assay for HIV-1 using a non-competitive internal standard in quantitative RT-PCR was coupled with real-time detection of both analyte and internal standard (IS) signals in a closed system. Real-time detection by the PE-ABI Prism 7700 relied on TaqMan probes specific for HIV and IS. The exogenous, non-competitive IS RNA was added in the same, known amount to a series of HIV RNA standards. The threshold cycle ratio from this internal standard calibration curve was used in the quantitation of HIV. Two configurations of reporter labels were compared. The HEX-HIV:FAM-IS system was the most precise, with nearly half-log discrimination over a range of 102 through 105 copies HIV-1 RNA. The FAM- HIV:HEX-IS system was less precise, but more sensitive and resistant to sample inhibition. The analysis of these signals and their impact on the range and precision of HIV quantitation is discussed. The design and synthesis of the fluorescently-labelled probes is also described.© (1998) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The inducible regulation of heat shock gene transcription is mediated by a family of heat shock factors (HSF) that respond to diverse forms of physiological and environmental stress including elevated temperature, amino acid analogs, heavy metals, oxidative stress, anti-inflammatory drugs, arachidonic acid, and a number of pathophysiological disease states. The vertebrate genome encodes a family of HSFs which are expressed ubiquitously, yet the DNA binding properties of each factor are negatively regulated and activated in response to specific conditions. This chapter will discuss the regulation of the HSF multi-gene family and the role of these transcriptional activators in the inducible expression of genes encoding heat shock proteins and molecular chaperones.

The heat shock response represents one of the most dramatic changes in gene expression and has served as a paradigm for inducible transcriptional responses. The response to temperature elevation, exposure to toxic agents, or other physiological stresses is universal and mediated through the induction of a highly conserved set of genes referred to as heat shock (HS) genes (Lindquist, 1986; Lindquist and Craig, 1988; Morimoto and Milarski, 1990; Morimoto, 1993; Morimoto et al, 1994). Studies in the early 1970’s had revealed that the elevation of temperature induces the synthesis of new polypeptides. It had been recognized early that these newly synthesized HS proteins (HSPs) are important to the survival response mounted by the cell. Perhaps the most significant effect of HS is on transcription. As the severity of the HS increases, the transcription of most genes is repressed and the genes coding for HSPs are transcriptionally induced 50 to 100-fold within minutes. HS has additional effects on mRNA stability and translational control which contribute to the preferential expression of HSPs (Lindquist, 1980; Storti et al, 1980; Banerji et al, 1984; Lindquist and Craig, 1988).

Multiple heat shock transcription factors (HSFs) have been discovered in several higher eukaryotes, raising questions about their respective functions in the cellular stress response. Previously, we had demonstrated that the two mouse HSFs (mHSF1 and mHSF2) interacted differently with the HSP70 heat shock element (HSE). To further address the issues of cooperativity and the interaction of multiple HSFs with the HSE, we selected new mHSF1 and mHSF2 DNA-binding sites through protein binding and PCR amplification. The selected sequences, isolated from a random population, were composed primarily of alternating inverted arrays of the pentameric consensus 5'-nGAAn-3', and the nucleotides flanking the core GAA motif were nonrandom. The average number of pentamers selected in each binding site was four to five for mHSF1 and two to three for mHSF2, suggesting differences in the potential for cooperative interactions between adjacent trimers. Our comparison of mHSF1 and mHSF2 binding to selected sequences further substantiated these differences in cooperativity as mHSF1, unlike mHSF2, was able to bind to extended HSE sequences, confirming previous observations on the HSP70 HSE. Certain selected sequences that exhibited preferential binding of mHSF1 or mHSF2 were mutagenized, and these studies demonstrated that the affinity of an HSE for a particular HSF and the extent of HSF interaction could be altered by single base substitutions. The domain of mHSF1 utilized for cooperative interactions was transferable, as chimeric mHSF1/mHSF2 proteins demonstrated that sequences within or adjacent to the mHSF1 DNA-binding domain were responsible. We have demonstrated that HSEs can have a greater affinity for a specific HSF and that in mice, mHSF1 utilizes a higher degree of cooperativity in DNA binding. This suggests two ways in which cells have developed to regulate the activity of closely related transcription factors: developing the ability to fully occupy the target binding site and alteration of the target site to favor interaction with a specific factor.

We studied the formation of linked circular DNA molecules promoted by the combined action of recl protein and type I topoisomerase of Ustilago maydis. When ATP was added as cofactor to reactions containing recl protein, pairs of homologous circular DNA molecules became linked after addition of topoisomerase. Closed circular duplex molecules could be joined at homologous sites with circular single-stranded molecules or with other circular duplex molecules, provided that homologous single-stranded DNA fragments or RNA polymerase and nucleoside triphosphates were also added. Complexes formed were topologically linked through regions of heteroduplex DNA. When the analog adenylyl-imidodiphosphate was substituted for ATP, nonhomologous pairs of circular DNA molecules became linked.

Progress in understanding the basis of recombination has lagged behind the related and overlapping areas of replication and repair for a number of reasons. For instance, the most favorable organisms for genetic analysis of recombination have not coincided with those most suitable for molecular studies. E. coli and its phages have been the prime source of molecular information, but some fairly obscure fungi have been instrumental in providing crucial genetic information about recombination. Furthermore, the phages of E. coli that were used most extensively for analysis of recombination were so different in their life cycles that it was not at all clear, at least in the beginning, that conclusions from any one of them could be generalized. Finally, recombination has not lent itself easily to dissection by biochemists because for a long time nobody could think of an adequate way to study the process. The problem was, if essentially identical chromosomes were crossed, what biochemical property could be exploited to measure the progress of the reaction?

Topoisomerase I cleavage sites have been mapped in vivo on the Hsp70 heat shock gene of Drosophila melanogaster cells using the drug camptothecin. Topoisomerase I cleavage was only observed when the Hsp70 gene was transcriptionally active. Site-specific single-strand DNA cleavage by topoisomerase I was confined to the transcribed region of the Hsp70 gene and occurred on both the transcribed and nontranscribed DNA strands. A number of the single-strand breaks on the complementary DNA strands occurred in close proximity giving rise to double-stranded DNA breaks. Inhibition of heat-induced Hsp70 transcription by either Actinomycin D (Act D) or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibited topoisomerase I cleavage except at the 5' and to a lesser extent the 3' end of the gene. Camptothecin (100 microM) inhibited transcription of the Hsp70 gene greater than 95%. These results suggest that topoisomerase I is intimately associated with and has an integral part in Hsp70 gene transcription.