Histone methyltransferase DOT1L coordinates AR and MYC stability in prostate cancer.

The histone methyltransferase DOT1L methylates lysine 79 (K79) on histone H3 and is involved in Mixed Lineage Leukemia (MLL) fusion leukemogenesis; however, its role in prostate cancer (PCa) is undefined. Here we show that DOT1L is overexpressed in PCa and is associated with poor outcome. Genetic and chemical inhibition of DOT1L selectively impaired the viability of androgen receptor (AR)-positive PCa cells and organoids, including castration-resistant and enzalutamide-resistant cells. The sensitivity of AR-positive cells is due to a distal K79 methylation-marked enhancer in the MYC gene bound by AR and DOT1L not present in AR-negative cells. DOT1L inhibition leads to reduced MYC expression and upregulation of MYC-regulated E3 ubiquitin ligases HECTD4 and MYCBP2, which promote AR and MYC degradation. This leads to further repression of MYC in a negative feed forward manner. Thus DOT1L selectively regulates the tumorigenicity of AR-positive prostate cancer cells and is a promising therapeutic target for PCa.

Ligand-activated peroxisome proliferator-activated receptor ß/δ modulates human endometrial cancer cell survival.

Endometrial cancer is the fourth most common malignancy among women and is a major cause of morbidity contributing to approximately 8,200 annual deaths in the USA. Despite advances to the understanding of endometrial cancer, novel interventions for the disease are necessary given that many tumors become refractory to therapy. As a strategy to identify novel therapies for endometrial carcinoma, in this study, we examined the contribution of the peroxisome proliferator-activated receptor ß/δ (PPARß/δ) to endometrial cancer cell proliferation and apoptosis. We found that when activated with the highly selective PPARß/δ agonists, GW0742 and GW501516, PPARß/δ inhibited the proliferation and markedly induced the apoptosis of three endometrial cancer cell lines. The specificity of the PPARß/δ-induced effects on cell proliferation and apoptosis was demonstrated using PPARß/δ-selective antagonists and PPARß/δ small interfering RNA in combination with PPARß/δ-selective agonists. Furthermore, we showed that PPARß/δ activation increased phosphatase and tensin homolog expression, which led to protein kinase B (AKT) and glycogen synthase kinase-3ß (GSK3ß) dephosphorylation, and increased ß-catenin phosphorylation associated with its degradation. Overall, our data suggest that the antitumorigenic effect of PPARß/δ activation in endometrial cancer is mediated through the negative regulation of the AKT/GSK3ß/ß-catenin pathway. These findings warrant further investigation of PPARß/δ as a therapeutic target in endometrial cancer.

Initiation of cancer and other diseases by catechol ortho-quinones: a unifying mechanism.

Exposure to estrogens is a risk factor for breast and other human cancers. Initiation of breast, prostate and other cancers has been hypothesized to result from reaction of specific estrogen metabolites, catechol estrogen-3,4-quinones, with DNA to form depurinating adducts at the N-7 of guanine and N-3 of adenine by 1,4-Michael addition. The catechol of the carcinogenic synthetic estrogen hexestrol, a hydrogenated derivative of diethylstilbestrol, is metabolized to its quinone, which reacts with DNA to form depurinating adducts at the N-7 of guanine and N-3 of adenine. The catecholamine dopamine and the metabolite catechol (1,2-dihydroxybenzene) of the leukemogen benzene can also be oxidized to their quinones, which react with DNA to form predominantly analogous depurinating adducts. Apurinic sites formed by depurinating adducts are converted into tumor-initiating mutations by error-prone repair. These mutations could initiate cancer by estrogens and benzene, and Parkinson's disease by the neurotransmitter dopamine. These data suggest a unifying molecular mechanism of initiation for many cancers and neurodegenerative diseases and lay the groundwork for designing strategies to assess risk and prevent these diseases.

Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene.

Treatment of SENCAR mouse skin with dibenzo[a,l]pyrene results in abundant formation of abasic sites that undergo error-prone excision repair, forming oncogenic H-ras mutations in the early preneoplastic period. To examine whether the abundance of abasic sites causes repair infidelity, we treated SENCAR mouse skin with estradiol-3,4-quinone (E(2)-3,4-Q) and determined adduct levels 1 h after treatment, as well as mutation spectra in the H-ras gene between 6 h and 3 days after treatment. E(2)-3,4-Q formed predominantly (> or =99%) the rapidly-depurinating 4-hydroxy estradiol (4-OHE(2))-1-N3Ade adduct and the slower-depurinating 4-OHE(2)-1-N7Gua adduct. Between 6 h and 3 days, E(2)-3,4-Q induced abundant A to G mutations in H-ras DNA, frequently in the context of a 3'-G residue. Using a T.G-DNA glycosylase (TDG)-PCR assay, we determined that the early A to G mutations (6 and 12 h) were in the form of G.T heteroduplexes, suggesting misrepair at A-specific depurination sites. Since G-specific mutations were infrequent in the spectra, it appears that the slow rate of depurination of the N7Gua adducts during active repair may not generate a threshold level of G-specific abasic sites to affect repair fidelity. These results also suggest that E(2)-3,4-Q, a suspected endogenous carcinogen, is a genotoxic compound and could cause mutations.

Regulation of histone acetylation and transcription by INHAT, a human cellular complex containing the set oncoprotein.

Acetylation of histones by p300/CBP and PCAF is considered to be a critical step in transcriptional regulation. In order to understand the role of cellular activities that modulate histone acetylation and transcription, we have purified and characterized a multiprotein cellular complex that potently inhibits the histone acetyltransferase activity of p300/CBP and PCAF. We have mapped a novel acetyltransferase-inhibitory domain of this INHAT (inhibitor of acetyltransferases) complex that binds to histones and masks them from being acetyltransferase substrates. Endogenous INHAT subunits, which include the Set/TAF-Ibeta oncoprotein, associate with chromatin in vivo and can block coactivatormediated transcription when transfected in cells. We propose that histone masking by INHAT plays a regulatory role in chromatin modification and serves as a novel mechanism of transcriptional regulation.

Evidence that error-prone DNA repair converts dibenzo[a,l]pyrene-induced depurinating lesions into mutations: formation, clonal proliferation and regression of initiated cells carrying H-ras oncogene mutations in early preneoplasia.

Initiation of skin tumors in mice is associated with the formation of oncogenic mutations in the H-ras gene. Mice treated on the dorsal skin with the potent polycyclic aromatic hydrocarbon (PAH) carcinogen dibenzo[a,l]pyrene (DB[a,l]P) form papillomas carrying the H-ras codon 61 (CAA to CTA) mutations. These mutations are induced in early preneoplastic skin within 1 day after DB[a,l]P treatment (Oncogene 16 (1998) 3203-3210) and appear to be related to DB[a,l]P-Ade-depurinating adducts (Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 10422-10426). The rapid kinetics of mutation induction suggests that abasic sites generated from base depurination may undergo error-prone excision repair in pre-S-phase cells to induce these mutations. Analysis of mutations in the H-ras exon 1 and 2 region in DB[a,l]P-treated early preneoplastic skin indicated great changes in mutation spectra in the preneoplastic period. The initial spectra contained abundant A-->G mutations, which frequently occurred 3' to a putative conserved sequence (TGN-doublet). These mutations appeared to be induced initially as mismatched (G.T) heteroduplexes and then converted into double-stranded mutations by one round of replication. Unlike the A-->G mutations found in DB[a, l]P-treated skin (which forms 99% depurinating adducts), A-->G mutations found in anti-DB[a,l]P-diol epoxide-treated skin (forms 97% stable adducts) did not appear to be G.T heteroduplexes. These results, therefore, suggest that under these conditions, the repair errors occurred only from abasic sites but not from stable adducts. Initiated cells carrying specific oncogenic mutations, formed presumably by misrepair, underwent rapid clonal expansion and regression (transient clonoplasia). The multiplication of initiated stem cells during transient clonoplasia may be a factor determining the tumor-initiating potential of some PAH carcinogens.

A viral mechanism for inhibition of p300 and PCAF acetyltransferase activity.

Nucleosomal histone modification is believed to be a critical step in the activation of RNA polymerase II-dependent transcription. p300/CBP and PCAF histone acetyltransferases (HATs) are coactivators for several transcription factors, including nuclear hormone receptors, p53, and Stat1alpha, and participate in transcription by forming an activation complex and by promoting histone acetylation. The adenoviral E1A oncoprotein represses transcriptional signaling by binding to p300/CBP and displacing PCAF and p/CIP proteins from the complex. Here, we show that E1A directly represses the HAT activity of both p300/CBP and PCAF in vitro and p300-dependent transcription in vivo. Additionally, E1A inhibits nucleosomal histone modifications by the PCAF complex and blocks p53 acetylation. These results demonstrate the modulation of HAT activity as a novel mechanism of transcriptional regulation.

Isolation and characterization of two proteins from Moraxella catarrhalis that bear a common epitope.

The UspA1 and UspA2 proteins of Moraxella catarrhalis are potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350, 000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100 degreesC. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

Linear amplification mapping of polycyclic aromatic hydrocarbon-reactive sequences in H-ras gene.

Linear amplification, or primer directed single-strand DNA synthesis, is commonly used in applications such as cycle sequencing and mapping replication block sites in DNA. Although linear amplification reactions would be expected to synthesize full-length single-stranded DNA, the synthesis is often prematurely terminated. We describe the optimization of a linear amplification protocol for synthesizing a full-length (985-nt) single-stranded pBR322 segment. The enzyme activities of five DNA polymerases commonly used in PCR amplification, namely, AmpliTaq, Stoffel fragment, Tth, Pfu, and Vent, were tested either singly or in combination. The results indicate that the additive action of small amounts of proofreading DNA polymerases to a nick-translating polymerase is optimum for linear amplification. From these results, a linear amplification protocol was developed to map DNA synthesis-blocking sites generated by the reaction of (+/-) anti-benzo[a]pyrene-7,8-diol-9,10-epoxide, or anti- or syn-dibenzo[a,l]pyrene-9,10-diol-11,12-epoxide with H-ras DNA surrounding the oncogenic codon 61 region. The results indicate that the central A of H-ras codon 61 (CAA) reacts with these polycyclic aromatic hydrocarbons.

Detection of dibenzo[a,l]pyrene-induced H-ras codon 61 mutant genes in preneoplastic SENCAR mouse skin using a new PCR-RFLP method.

One of the key events in tumor initiation in mouse skin is mutational activation of the H-ras gene. Papillomas induced by the most carcinogenic environmental polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DB[a,l]P), in SENCAR mouse skin contain a specific H-ras codon 61 (CAA-->CTA) mutation. We describe here detection of these mutations in preneoplastic skin by measuring the frequency of an induced XbaI RFLP, created by the mutation. Development of the PCR-XbaI RFLP method, sensitive enough to detect 1 codon 61 mutant allele among 10,000 wild-type genes, is described. The results indicate that codon 61 mutations are induced 1 day (0.1%) after DB[a,l]P treatment on mouse skin, reach a high value (5%) by day 3, rapidly decline between days 7-9 and increase again during the clonal expansion of pre-papillomas into tumors. The detection of codon 61 mutations 1 day after DB[a,l]P exposure suggests that mutations occurred by pre-replication misrepair.

Purification and characterization of a phagocytosis inducing factor: role in cell-substratum adherence.

The presence of two different signaling molecules is essential to induce opsonin-independent phagocytosis of particulate activators of the human alternative complement pathway by human monocytes. In addition to the involvement of a low M(r) peptide cytokine or phagocytosis inducing factor (PIF), we have now established that the participation of bacterial lipopolysaccharide is also required. PIF has been demonstrated to be present in human cell lines of different origins, e.g., WISH cells, Raji cells, U937 cells, HL-60 cells and M21 cells. PIF has been purified to apparent homogeneity from the U937 cell line by anion-exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sephadex G-50. On the basis of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular mass has been estimated to be approximately 1,600 Da. PIF also plays an important role in the regulation of cell-substratum adherence.

Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300.

We report here the identification of a novel cofactor, ACTR, that directly binds nuclear receptors and stimulates their transcriptional activities in a hormone-dependent fashion. ACTR also recruits two other nuclear factors, CBP and P/CAF, and thus plays a central role in creating a multisubunit coactivator complex. In addition, and unexpectedly, we show that purified ACTR is a potent histone acetyltransferase and appears to define a distinct evolutionary branch to this recently described family. Thus, hormonal activation by nuclear receptors involves the mutual recruitment of at least three classes of histone acetyltransferases that may act cooperatively as an enzymatic unit to reverse the effects of histone deacetylase shown to be part of the nuclear receptor corepressor complex.

Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase.

The transcriptional corepressors SMRT and N-CoR function as silencing mediators for retinoid and thyroid hormone receptors. Here we show that SMRT and N-CoR directly interact with mSin3A, a corepressor for the Mad-Max heterodimer and a homolog of the yeast global-transcriptional repressor Sin3p. In addition, we demonstrate that the recently characterized histone deacetylase 1 (HDAC1) interacts with Sin3A and SMRT to form a multisubunit repressor complex. Consistent with this model, we find that HDAC inhibitors synergize with retinoic acid to stimulate hormone-responsive genes and differentiation of myeloid leukemia (HL-60) cells. This work establishes a convergence of repression pathways for bHLH-Zip proteins and nuclear receptors and suggests this type of regulation may be more widely conserved than previously suspected.

Two contact regions between Stat1 and CBP/p300 in interferon gamma signaling.

Interferon gamma (IFN-gamma) induces rapid tyrosine phosphorylation of the latent cytoplasmic transcription factor, Stat1, which then forms homodimers, translocates to the nucleus and participates in IFN-gamma-induced transcription. However, little is known of the interactions between Stat1 and the general transcription machinery during transcriptional activation. We show here that Stat1 can directly interact with the CREB-binding protein (CBP)/p300 family of transcriptional coactivators. Specifically, two interaction regions were identified: the amino-terminal region of Stat1 interacts with the CREB-binding domain of CBP/p300 and the carboxyl-terminal region of Stat1 interacts with the domain of CBP/p300 that binds adenovirus E1A protein. Transfection experiments suggest a role for these interactions in IFN-gamma-induced transcription. Because CBP/p300-binding is required for the adenovirus E1A protein to regulate transcription of many genes during viral replication and cellular transformation, it is possible that the anti-viral effect of IFN-gamma is based at least in part on direct competition by nuclear Stat1 with E1A for CBP/p300 binding.

In vivo molecular analysis of cytokines in a murine model of ocular onchocerciasis. I. Up-regulation of IL-4 and IL-5 mRNAs and not IL-2 and IFN gamma mRNAs in the cornea due to experimental interstitial keratitis.

Sclerosing keratitis is the major cause of blindness due to onchocerciasis which results from chronic infection with the filarial parasite Onchocerca volvulus. Using a murine model of onchocercal sclerosing keratitis, we have demonstrated previously that predominantly (> 85%) CD3 + /CD4+ T-cells as well as the IL-2 receptor bearing cells infiltrate into the cornea in vivo during development and progress of the disease. The identification of CD4+ subsets TH1 and TH2 based on the cytokine secretion patterns of murine T-lymphocytes has been useful for understanding the immune basis of resistance and pathogenesis in murine models of several parasitic diseases. The present investigation was carried out to demonstrate whether the local immune response at the corneal lesion due to onchocercal interstitial keratitis correlated with such distinct patterns of cytokine production. For that purpose, mRNA was extracted separately from corneas obtained from the diseased eyes and the normal eyes of A/J mice with onchocercal interstitial keratitis, reverse transcribed and amplified by the polymerase chain reaction with four different cytokine specific primers. In corneas obtained from the eyes affected with onchocercal interstitial keratitis, mRNAs coding for IL-4 and IL-5 were up-regulated compared to the normal eyes having no lesions from the same animals. However, the levels of mRNAs for IL-2 and IFN gamma were found to be the same in the diseased and normal eyes. Taken together, these data suggest that IL-4 and IL-5 producing TH2-lymphocytes are active at the corneal lesion due to onchocercal interstitial keratitis.

Role of CBP/P300 in nuclear receptor signalling.

The nuclear receptor superfamily includes receptors for steroids, retinoids, thyroid hormone and vitamin D, as well as many related proteins. An important feature of the action of the lipophilic hormones and vitamins is that the maintenance of homeostatic function requires both intrinsic positive and negative regulation. Here we provide in vitro and in vivo evidence that identifies the CREB-binding protein (CBP) and its homologue P300 (refs 6,7) as cofactors mediating nuclear-receptor-activated gene transcription. The role of CBP/P300 in the transcriptional response to cyclic AMP, phorbol esters, serum, the lipophilic hormones and as the target of the E1A oncoprotein suggests they may serve as integrators of extracellular and intracellular signalling pathways.

Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: the role of apurinic sites.

Mouse skin tumors contain activated c-H-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2. Mutagenesis by the noncoding apurinic sites can produce G-->T and A-->T transversions by DNA misreplication with more frequent insertion of deoxyadenosine opposite the apurinic site. Papillomas were induced in mouse skin by several aromatic hydrocarbons, and mutations in the c-H-ras gene were determined to elucidate the relationship among DNA adducts, apurinic sites, and ras oncogene mutations. Dibenzo[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, anti-DB[a,l]P-11,12-diol-13,14-epoxide, DB[a,l]P-8,9-dihydrodiol, 7,12-dimethylbenz[a]anthracene (DMBA), and 1,2,3,4-tetrahydro-DMBA consistently induced a CAA-->CTA mutation in codon 61 of the c-H-ras oncogene. Benzo[a]pyrene induced a GGC-->GTC mutation in codon 13 in 54% of tumors and a CAA-->CTA mutation in codon 61 in 15%. The pattern of mutations induced by each hydrocarbon correlated with its profile of DNA adducts. For example, both DB[a,l]P and DMBA primarily form DNA adducts at the N-3 and/or N-7 of deoxyadenosine that are lost from the DNA by depurination, generating apurinic sites. Thus, these results support the hypothesis that misreplication of unrepaired apurinic sites generated by loss of hydrocarbon-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin.

Isolation and immunochemical characterization of eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae.

Eukaryotic translation initiation factor 5 (eIF-5), which catalyzes the hydrolysis of GTP bound to the 40 S ribosomal initiation complex has been purified from yeast cell lysates. The purified factor eluted from gel filtration columns as a protein of apparent M(r) = 45,000-50,000. However, when the purified preparation was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two distinct polypeptides of apparent M(r) = 54,000 and 56,000 were observed. Each of the two polypeptides individually was found to contain eIF-5 activity, and they were immunologically related to each other. In less pure preparations of yeast eIF-5, however, a significant proportion of eIF-5 activity eluted from gel filtration columns as a protein of M(r) > 140,000. Immunochemical methods were therefore employed to determine the molecular structure of eIF-5 in crude yeast cell lysates. Antisera against purified yeast eIF-5 were prepared in rabbits and shown to be highly potent in inhibiting eIF-5-mediated 80 S initiation complex formation. When crude eIF-5 preparations, as well as yeast cells that were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate, were analyzed by Western blots probed with affinity-purified anti-eIF-5 antibodies, a major immunoreactive polypeptide (apparent M(r) = 54,000) and a minor band (apparent M(r) = 56,000) were observed. No precursor forms of molecular weight higher than 56,000 were detected in any preparations. These results suggest that yeast eIF-5 is a monomeric protein of apparent M(r) = 50,000-56,000.

Structural homology of complement protein C6 with other channel-forming proteins of complement.

The amino acid sequence of the amino-terminal half of the complement protein C6 has been found to show overall structural homology with the homologous regions of the channel-forming proteins C7, C8 alpha, C8 beta, and C9. In addition, two specific cysteine-rich segments common to the amino-terminal regions of C7, C8 alpha, C8 beta, and C9 also occur in their expected positions in C6, suggesting functional significance. Two cDNA clones encoding C6 were isolated from a human liver library in the bacteriophage vector lambda gt11. The predicted protein sequence contains an apparent initiation methionine and a putative signal peptide of 21 residues, as well as a site for N-glycosylation at residue 303. The sequence of the C6 protein reported here has 47-52% similarity with C7, C8 alpha, C8 beta, and C9, as well as 31-38% similarity with thrombospondin, thrombomodulin, and low density lipoprotein receptor. The sequence data have been interpreted by using computer algorithms for estimation of average hydrophobicity and secondary structure.

The structure of human complement component C7 and the C5b-7 complex.

The molecular architecture of human complement component C7 was elucidated at several structural levels. The complete primary structure of C7 was derived from the cDNA sequence of clones isolated from a human liver library. C7 is a mosaic protein that consists of 821 amino acids. The amino-terminal two-thirds of C7 has 23-30% homology with complement components C8 and C9. In addition, the carboxyl-terminal third contains four cysteine-rich segments that have overlapping internal homology. The protein is a single polypeptide chain with 28 disulfide bonds and is glycosylated at two sites. Virtually all the cysteines are found in small units of 35-77 amino acids that exhibit homology with those of various proteins including the low density lipoprotein receptor, epidermal growth factor precursor, thrombospondin, and blood coagulation factors IX and X. The secondary structural analysis, estimated by circular dichroism, suggested a high content of beta-sheet (38%) and beta-turns (24%). The tertiary structure, visualized by transmission electron microscopy, indicated a flexible elongated molecule with dimensions of 151 X 59 X 43 A. The quaternary structure of the C5b-7 complex bound to lipid vesicles was observed to be in the form of monomers or dimers. The monomer C5b-7 consists of a leaflet and a long flexible stalk, and the dimer has two leaflets linked through a supercoiled stalk. Membrane binding is mediated by the stalk part of the complexes. Using a radioiodinated photoreactive cross-linking reagent bound to the polar head group of phosphatidylethanolamine, the stalk part of the C5b-7 complex could be labeled preferentially, and it was found to consist mainly of C6 and C7. Thus, C7 plays a major role in bringing about the hydrophilic-amphiphilic transition during the formation of the membrane attack complex, and it serves as a membrane anchor for the C5b-7 complex.