David E. Mold

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.

Reversal of multidrug resistance by two nordihydroguaiaretic acid derivatives, M4N and maltose-M3N, and their use in combination with doxorubicin or paclitaxel.

Purpose: Multidrug resistance (MDR) continues to be a major obstacle for successful anticancer therapy. One of the principal factors implicated in MDR is the over expression of P-glycoprotein (Pgp), the product of the MDR1 gene. Methods: Here we explore the possibility of using the transcription inhibitor tetra-O-methyl nordihydroguaiaretic acid (M4N) to inhibit Sp1-regulated MDR1 gene expression and restore doxorubicin and paclitaxel sensitivity to multidrug resistant human cancer cells in vitro and in vivo. Results: We found that M4N acted synergistically with doxorubicin and paclitaxel in inhibiting the growth of the cells in culture allowing significant dose reductions of both drugs. We observed no such synergism when M4N was used in combination with cisplatin, another chemotherapeutic agent, but not a Pgp substrate, as analyzed by the combination index and isobologram methods. Analysis of MDR1 mRNA and Pgp levels revealed that at sublethal doses, M4N inhibited MDR1 gene expression in the multidrug resistant NCI/ADR-RES cells and reversed the MDR phenotype as measured by Rhodamine-123 retention. In addition, M4N was found to inhibit doxorubicin-induced MDR1 gene expression in drug sensitive MCF-7 breast cancer cells. Conclusions: M4N and maltose-tri-O-methyl nordihydroguaiaretic acid (maltose-M3N), a water-soluble derivative of NDGA, were also able to reverse the MDR phenotype of the tumor cells in a xenograft model system and combination therapy with M4N or maltose-M3N and paclitaxel was effective at inhibiting growth of these tumors in nude mice.

Inhibition of HSV-1 replication and reactivation by the mutation-insensitive transcription inhibitor tetra-O-glycyl-nordihydroguaiaretic acid

Methylated derivatives of nordihydroguaiaretic acid (NDGA)were previously shown to be potent mutation-resistant inhibitors of herpes simplex virus type 1 (HSV-1) which target Sp1 protein binding to critical viral promoters. The hydrophobic nature of these agents, however, renders them relatively water-insoluble and, therefore, limits their applicability. We report here on the anti-HSV-1 properties of a related but water-soluble glycylated derivative of NDGA, tetra-O-glycyl-NDGA (G(4)N). In yield reduction assays, G(4)N inhibited replication of laboratory and clinical strains of wild type HSV-1 and ACV-resistant (HSV-1(R)) strains of HSV-1 in a dose-dependent manner, with average IC(50) values of 4.7 and 3.2 microM against wild-type and HSV-1(R) strains, respectively. An MTT-based cytotoxicity assay revealed a TC(50) value of 73.2 microM for G(4)N on Vero cells, with no reduction in viability detected at concentrations below 30 microM. Similar to its methylated counterparts, G(4)N was found to inhibit transcription of the HSV-1 ICP4 gene, a major immediate early viral regulator, and gel mobility shift assays showed it can block Sp1 protein binding to cognate sites on the ICP4 promoter. In anticipation of its potential use as a systemic anti-HSV-1 agent, we tested G(4)N in a murine trigeminal ganglia (TG) explant model system, and found G(4)N was able to prevent HSV-1 reactivation from explanted and cultured latently infected TG.

Regulation of Zebrafish Zona Pellucida Gene Activity in Developing Oocytes

Abstract The two major zona pellucida proteins of the zebrafish chorion, Zp2 and Zp3, are encoded by multicopy genes arranged in tandem arrays on chromosomes 20 and 2, respectively. Expression of these zp genes in zebrafish is oocyte specific, and we report herein that their activity in developing oocytes is dependent on conserved CCAAT box sites in their promoters. A 140-bp region immediately upstream of the transcription initiation site (position 1) of the zp2 genes has been homogenized by gene conversion and contains a single CCAAT box located at −138 that is necessary for promoter activity in oocytes residing in stage I and early stage II ovarian follicles as determined by microinjection of promoter constructs linked to a luciferase reporter gene. The zp3 gene promoters have two inverted CCAAT boxes located in a region of shared homology within the initial 175 nucleotides. Serial deletion of these sites resulted in incremental decreases in luciferase activity. Double-stranded oligonucleotides containing CCAAT box sequences from both genes formed CCAAT box-specific complexes with ovarian follicle extracts in an electrophoretic mobility shift assay. We also found that the expression of the separate zebrafish zp3b gene, more closely related to two oocyte-expressed medaka zpc genes than to the tandemly arrayed zebrafish zp3 genes, is not CCAAT box dependent. The significance that these results have in furthering our understanding of the regulation of zebrafish zp gene evolution and regulation is discussed.

In vivo amelioration of endogenous antitumor autoantibodies via low-dose P4N through the LTA4H/activin A/BAFF pathway

Significance This study finds that a small-molecule drug (P4N) is able to inhibit tumor growth by augmentation of endogenous antitumor autoantibodies (EAAs). We show that the enhancement of EAA activity by P4N is mediated through activation of the leukotriene A4 hydrolase (LTA4H)/activin A/B-cell activating factor (BAFF) pathway, revealing a valuable method for developing new immune modulators of tumor growth via humoral immunity. Typically, the effects of the humoral response on tumor inhibition are modest; however, the results of this study demonstrate that by removing the impediment to cancer cell destruction posed by low-activity autoantibodies, the realization of new, more potent immunotherapies for cancer treatment may be possible. Cancer progression is associated with the development of antitumor autoantibodies in patients’ sera. Although passive treatment with antitumor antibodies has exhibited remarkable therapeutic efficacy, inhibitory effects on tumor progression by endogenous antitumor autoantibodies (EAAs) have been limited. In this study, we show that P4N, a derivative of the plant lignan nordihydroguaiaretic acid (NDGA), enhanced the production of EAAs and inhibited tumor growth at low noncytotoxic concentrations via its immunoregulatory activity. Intratumoral injection of P4N improved the quantity and quality of EAAs, and passive transfer of P4N-induced EAAs dramatically suppressed lung metastasis formation and prolonged the survival of mice inoculated with metastatic CT26 tumor cells. P4N-induced EAAs specifically recognized two surface antigens, 78-kDa glucose-regulated protein (GRP78) and F1F0 ATP synthase, on the plasma membrane of cancer cells. Additionally, P4N treatment led to B-cell proliferation, differentiation to plasma cells, and high titers of autoantibody production. By serial induction of autocrine and paracrine signals in monocytes, P4N increased B-cell proliferation and antibody production via the leukotriene A4 hydrolase (LTA4H)/activin A/B-cell activating factor (BAFF) pathway. This mechanism provides a useful platform for studying and seeking a novel immunomodulator that can be applied in targeting therapy by improving the quantity and quality of the EAAs.

Hepatocellular Carcinoma Targeting Agents: Conjugates of Nitroimidazoles with Trimethyl Nordihydroguaiaretic Acid

Cancer has been a primary global health issue for decades, with hepatocellular carcinoma (HCC) resulting in more than half a million new cases annually. With survival rates as low as <5 % after five years, it remains a poorly treated cancer. Nordihydroguaiaretic acid (NDGA), an antioxidant, was previously proven effective against cancer cells. Nitroimidazole derivatives convert into reactive compounds under hypoxic conditions. In this study, eight methylated NDGAs containing a 2‐ or 4‐nitroimidazole moiety were synthesized as leads against HCC. Four of these conjugates, possessing a poly(ethylene glycol) tether, had superior aqueous solubility. These four NDGA–nitroimidazole conjugates were found to inhibit the proliferation HCC Hep3B cells with IC50 values between 10 and 15 μM. Furthermore, nitroimidazole‐conjugated NDGA derivatives exhibit better antiproliferative activity under hypoxic conditions.

Cell lineage-specific expression of the MIPP gene

The expression of the mouse IAP promoted placental (MIPP) gene was examined in the tissues of the 13.5 day mouse conceptus by Northern hybridization analysis. MIPP transcripts were found in total cellular RNA isolated from the placenta, parietal yolk sac, and visceral yolk sac, but not the embryo or amnion. Expression in the visceral yolk sac was confined to the visceral endoderm cell layer suggesting that the MIPP gene is specifically expressed in tissues derived from the trophectoderm and primitive endoderm cell lineages. The expression of IAP genes paralleled that of MIPP in all tissues studied except for the presence of IAP transcripts in the amnion.