Ru Chih C. Huang

Effect of protein-bound RNA associated with chick embryo chromatin on template specificity of the chromatin

Abstract The possible function of the protein-bound RNA in chick embryo chromatin is examined by experiments involving chromatin reconstitution and DNA-RNA hybridization of the template products. In the presence of this RNA, functional chromatin may be reconstructed from the dissociated chromatin by gradient dialysis from high to low salt in the presence of urea. RNAs, synthesized in vitro using such a reconstituted chromatin as a template, resemble the native RNA isolated from nuclei for their ability to form specific molecular hybrids with DNA. Degradation of this RNA by zinc nitrate resulted in a diminishing of this specific reassociation. RNAs primed by zinc-treated chromatin differ from either nuclear RNA or the original chromatin-primed RNA. Although protein-bound RNA is found in native chromatin, the origin of its coupling (protein to RNA) is unknown at the present time.

Inhibition of human papillomavirus type 16 gene expression by nordihydroguaiaretic acid plant lignan derivatives

Several methylated derivatives of a plant lignan, nordihydroguaiaretic acid (NDGA) were found to be potent anti-viral agents by suppressing Sp1 regulated transcription within the sexually transmitted viruses human immunodeficiency virus (HIV) and herpes simplex virus (HSV). A prominent Sp1 DNA binding site within many human papillomavirus (HPV) promoters has been noted to play an active role in HPV gene expression. In this report it is shown that the three NDGA derivatives, Mal.4, M(4)N, and tetra-acetyl NDGA can also inhibit gene expression from the early promoter P(97) of HPV16. The drug activity on gene expression was measured after DNA transfection of recombinant vector constructs linking the viral promoter and enhancer elements to the luciferase reporter gene. Using the specific luciferase activity as the indicator of gene expression, Mal.4 and M(4)N were found to be active in a dose dependent manner that is in the same range of concentrations reported for the promoters of HIV, HSV, and simian virus 40 (SV40) while tetra-acetyl NDGA was much more active in suppression of the HPV P(97) promoter activity than Mal.4 and M(4)N. The drugs showed limited to no effect on gene expression driven by the adenovirus major late promoter and the cytomegalovirus (CMV) promoter. Hence, such drug derivatives may be significant in the therapy of papillomavirus infections and their associated induced human cancers.

Binding of actinomycin D to calf thymus chromatin.

Abstract Studies are made on the binding of actinomycin D to calf thymus DNA and chromatin. The effect upon binding of the removal of chromosomal protein by NaCl is examined. Protein is extracted from chromatin with 0.6, 1.0 and 2.0 m -NaCl, and is separated from the remaining nucleoprotein on a Bio-gel A50 column. The amount of total protein, histone and acid-insoluble protein removed at different salt concentrations is examined, and the type of histone removed is determined using analytical disc electrophoresis. Ultraviolet absorption spectra and melting profiles of the nucleoprotein samples are also taken. Actinomycin D binding to chromatin, salt-extracted chromatin, and DNA is examined spectrophotometrically, and the resulting data are used to graph Scatchard plots, from which both binding constants and the number of actinomycin D molecules bound to DNA are determined. Chromatin, like DNA, appears to have more than one set of binding sites, and the set of strongest binding sites appears to be identical for DNA and chromatin. DNA has close to three times as many strong binding sites as chromatin. The number of actinomycin D molecules bound to these strong binding sites shows a non-linear relationship with the percentage of total protein and histone protein removed, rising more rapidly per unit amount of protein when the last portions of protein are removed. The removal with salt of chromosomal protein not extractable with 0.4 n -H 2 SO 4 , which contains some histone as well as non-histone protein, shows a more linear relationship with the increase in actinomycin D binding.

Reconstitution of chromatin. The sequential binding of histones to DNA in the presence of salt and urea.

Calf thymus chromatin is dissociated in 3 m-NaCl, 0.05 m-NaHSO3 and 6 m-urea, pH 7.8. The presence of NaHSO3 is necessary to prevent degradation of calf thymus histones. The protein is returned to the DNA by a stepwise dialysis into solutions of lower NaCl concentrations, maintaining constant urea and NaHSO3 concentrations. At a given NaCl concentration, free protein is separated from DNA-bound protein on a Bio-Gel A50 column equilibrated in the same salt and urea concentrations as the dialyzed sample. Both free and DNA-bound histones are analyzed by analytical disc-acrylamide gel electrophoresis. Urea lowers the affinity of histones for DNA. None of the histones return to the DNA until a NaCl concentration of 0.2 m has been reached. The first histone to bind is lysine-rich histone I. The other histones return to the DNA during dialysis from 0.15 m-NaCl, 0.05 m-NaHSO3 and 6 m-urea to 6 m-urea, which is followed by a final dialysis into 2 × 10−4 m-EDTA, pH 6.2. When native chromatin is extracted with NaCl in the presence of 6 m-urea and 0.04 m-NaHSO3, slightly lysine-rich histones IIb1 and IIb2 are removed at 0.15 m-NaCl, lysine-rich histone I at 0.3 m-NaCl and the arginine-rich histones III and IV at 0.6 m-NaCl. Increasing the urea concentration to 7 m lowers the NaCl concentration required to remove histones III and IV to 0.15 m-NaCl, but does not change the NaCl concentration required to remove histones I, IIb1 and IIb2. The NaCl concentration at which histones III and IV associate with DNA appears to be a function of the urea concentration and time used for extraction.

Isolation of anti-HIV-1 lignans from Larrea tridentata by counter-current chromatography

Several lignans, mostly new, were isolated from Larrea tridentata by assay-guided counter-current chromatography (CCC). Using the secreted alkaline phosphatase bioassay of HIV Tat transactivation and the two-phase hexane-ethyl acetate-methanol-water solvent system, two major components (Gr and Lo) were identified as anti-HIV active principles. The chemical structures of the constituents of Gr (G1-G4) and Lo (L1-L4) were determined by GC-MS and NMR. After optimization of isolation conditions, a large-scale isolation with the chloroform-methanol-water system yielded five constituents (FB1-FB5). The most predominant anti-HIV compound FB2 (denoted Malachi 4:5-6 or mal.4), which occurs in 0.23% yield, was separated from its FB1 isomer (0.13% yield). Compound FB4 and two tricyclic lignans (FB3 and FB5) were also isolated in a substantial amount for further testing of their anti-HIV activities. These compounds may represent a new class of anti-HIV agents with important clinical relevance.

Isolation of messenger rna for an immunoglobulin kappa chain and enumeration of the genes for the constant region of kappa chain in the mouse.

Abstract The messenger RNA for an immunoglobulin light (kappa) chain was isolated from the mouse myeloma MOPC41 and shown to be almost twofold longer than necessary to code for its protein product. DNA complementary to the mRNA was synthesized with the RNA-directed DNA polymerase of Rous sarcoma virus. Although the individual chains of the cDNA † contained an average of only 270 nucleotides, the cDNA was heterogeneous in molecular weight, allowing the isolation of a fraction of the cDNA 620 nucleotides long. This large cDNA would be long enough to code for nearly all (95%) of the constant region if all the untranslated region of the mRNA were between the 3′ terminal poly(A) and the constant region. On the other hand, if all the untranslated region of the mRNA were at the 5′ terminus, this cDNA would code for 93% of the entire kappa chain. The specificity of nucleotide sequences in the cDNA was documented by molecular hybridization with both template RNA and RNA from various myelomas. The amount of hybridization obtained with myeloma RNA was approximately proportional to the amount of kappa chain protein produced by the various myeloma cells. In addition, there was no hybridization with RNA isolated from either BALB/c mouse liver or Escherichia coli . The genes for the constant region of the kappa chain were enumerated in the mouse genome by annealing cDNA to DNA from mouse liver and MOPC41 myeloma. The haploid genome of both tissues contained three to four genes for the constant region of kappa chain even when tested under conditions that would detect distantly related nucleotide sequences. The fact that there are only a few genes coding for the constant region of kappa chains implies that specialized genetic mechanisms are required for the generation of antibody diversity.

Cluster of genes encoding the major egg envelope protein of zebrafish.

The proteins of fish egg envelopes are encoded by genes that are closely related to the genes for human zona pellucida proteins. A cluster of three genes coding for an egg envelope protein was isolated from the zebrafish, Danio rerio. The three genes, zp2a, zp2b, and zp2c, are located within an 11 kb region and are each comprised of eight exons spanning 1.85 kb. The exon–intron structures of the genes are nearly identical; however, their deduced amino acid sequences diverge at exon 7 (zp2b and zp2c from zp2a) and exon 8 (zp2c from zp2b). Exons 2–7 have a structural organization similar to exons in the carboxy‐terminal half of the human zona pellucida ZP1, ZP2, and ZPB genes, suggesting they arose from a common ancestral gene. Sequence comparisons indicate that the deduced zebrafish proteins are most closely related to human ZPB. Zebrafish mRNAs coding for each of the three ZP2 variants have been found either as full‐length cDNAs or expressed sequence tags. Distinct from the wf♀ gene of winter flounder which we first reported (Lyons et al., 1993 : J Biol Chem 268:21351–21358), expression of the zebrafish zp2 genes was found to be ovary‐specific, instead of liver‐specific, and the promoter regions of zp2a and zp2b, while different, both contained E‐box sequences (CANNTG) that have been demonstrated to be essential for coordination of zona pellucida gene expression in mammalian oocytes. Mixed peptide sequence analysis was used to identify the major polypeptide component of isolated zebrafish egg envelopes as the zp2 gene product. Mol. Reprod. Dev. 58:4–14, 2001.

Systemic Treatment with Tetra-O-Methyl Nordihydroguaiaretic Acid Suppresses the Growth of Human Xenograft Tumors

Purpose: We have previously shown that the transcriptional inhibitor tetra-O-methyl nordihydroguaiaretic acid (M4N) induces growth arrest in tumor cells and exhibits tumoricidal activity when injected intratumorally into tumor cell explants in mice. The experiments reported here were designed to determine whether M4N can be given systemically and inhibit the growth of five different human xenograft tumors. Experimental Design: Nude (nu/nu) mice bearing xenografts of each of five human tumor types (i.e., hepatocellular carcinoma, Hep 3B; prostate carcinoma, LNCaP; colorectal carcinoma, HT-29; breast carcinoma, MCF7; and erythroleukemia, K-562) were treated with M4N given i.v. or i.p. in a Cremophor EL–based solvent system or orally in a corn oil based diet. Tumors from the treated animals were measured weekly and analyzed for the expression of the Cdc2 and survivin genes, both previously shown to be down-regulated by M4N. Results: Systemic M4N treatment suppressed the in vivo growth of xenografts in each of the five human tumor types. Four of the five tumor models were particularly sensitive to M4N with tumor growth inhibitions (T/C values) of ≤42%, whereas the fifth, HT-29, responded to a lesser extent (48.3%). Growth arrest and apoptosis in both the xenograft tumors and in the tumor cells grown in culture were accompanied by reductions in both Cdc2 and tumor-specific survivin gene expression. Pharmacokinetic analysis following oral and i.v. administration to ICR mice indicated an absolute bioavailability for oral M4N of ∼88%. Minimal drug-related toxicity was observed. Conclusion: These preclinical studies establish that when given systemically, M4N can safely and effectively inhibit the growth of human tumors in nude mice.

Isolation and characterization of IPP, a novel human gene encoding an actin-binding, kelch-like protein

The kelch family of proteins is defined by a 50 amino-acid repeat that has been shown to associate with actin. Here we describe the cloning and initial characterization of IPP, a novel human gene that predicts a kelch family protein homologous to the mouse Ipp gene, a previously described kelch family member. A 3kb IPP cDNA clone was isolated from a human placenta library using a probe derived from Ipp. Restriction mapping and Southern blot analysis show that IPP comprises eight exons spanning more than 47kb of genomic DNA. Fluorescence in situ hybridization maps the gene to chromosome 1p32-1p34. Northern blot analysis reveals transcripts of 1.4, 2.2, 5. 0, and 7.3kb. The 1.4 and 2.2kb messages are found exclusively in testis, while the 5.0 and 7.3kb messages are expressed at varying levels in ovary, placenta, small intestine, spleen, testis, and thymus. The IPP cDNA clone contains a 1752bp open reading frame that predicts a 584 amino-acid, 66kDa protein. Sequence analysis indicates an N-terminal POZ protein-protein interaction domain and a C-terminal kelch repeat domain consisting of six tandemly arranged repeats. Cosedimentation assays performed with these domains expressed as glutathione S-transferase fusion proteins demonstrate an actin-binding activity mediated specifically by the kelch repeat domain of IPP.

CDC2/CDK1 expression in esophageal adenocarcinoma and precursor lesions serves as a diagnostic and cancer progression marker and potential novel drug target

Esophageal adenocarcinoma arises through well-defined precursor lesions (Barrett esophagus), although only a subset of these lesions advances to invasive adenocarcinoma. The lack of markers predicting progression in Barrett esophagus, typical presentation at advanced stage, and limitations of conventional chemotherapy result in >90% mortality for Barrett-associated adenocarcinomas. To identify potential prognostic markers and therapeutic targets, we compared gene expression profiles from Barrett-associated esophageal adenocarcinoma cell lines (BIC1, SEG1, KYAE, OE33) and normal esophageal epithelial scrapings utilizing the Affymetrix U133_A gene expression platform. We identified 560 transcripts with >3-fold up-regulation in the adenocarcinoma cell lines compared with normal epithelium. Utilizing tissue microarrays composed of normal esophageal squamous mucosa (n = 20), Barrett esophagus (n = 10), low-grade dysplasia (n = 14), high-grade dysplasia (n = 27), adenocarcinoma (n = 59), and node metastases (n = 27), we confirmed differential up-regulation of three proteins (Cdc2/Cdk1, Cdc5, and Igfbp3) in adenocarcinomas and Barrett lesions. Protein expression mirrored histologic progression; thus, 87% of low-grade dysplasias had at least focal surface Cdc2/Cdk1 and 20% had >5% surface staining; 96% of high-grade dysplasias expressed abundant surface Cdc2/Cdk1, while invasive adenocarcinoma and metastases demonstrated ubiquitous expression. Esophageal adenocarcinoma cell lines treated with the novel CDC2/CDK1 transcriptional inhibitor, tetra-O-methyl nordihydroguaiaretic acid (EM-1421, formerly named M4N) demonstrated a dose-dependent reduction in cell proliferation, paralleling down-regulation of CDC2/CDK1 transcript and protein levels. These findings suggest a role for CDC2/CDK1 in esophageal adenocarcinogenesis, both as a potential histopathologic marker of dysplasia and a putative treatment target.