Sesquiterpene antitumor agents: inhibitors of cellular metabolism.

Helenalin and tenulin injected into CF1 male mice bearing Ehrlich ascites tumors inhibit DNA synthesis and DNA polymerase enzymatic activity in the tumor cells. Helenalin inhibited protein synthesis. Both drugs increased the concentration of adenosine 3',5'-monophosphate, and interfered with glycolytic and mitochondrial energy processes. Cholesterol synthesis was also inhibited, resulting in lower serum cholesterol levels in tumor-bearing animals. Data obtained in vitro indicate that the cyclopentenone-bearing sesquiterpene lactone and related compounds do not alkylate puring bases of nucleic acids but rather undergo a Michael-type addition reaction with the sulfhydryl groups of reduced glutathione and l-cysteine. Thus, the inhibition of cellular enzyme activities and metabolism that has been observed with these drugs might be explained by the occurrence of a Michael-type teaction.

Human cytomegalovirus and herpes simplex type 2 virus in normal and adenocarcinomatous prostate glands.

Normal prostate, benign prostate hypertrophy (BHP), and prostate adenocarcinoma (ACP) biopsy specimens were analyzed for the presence of human cytomegalovirus (HCMV) and herpes simplex virus type 2 (HSV-2)-specific macromolecules. HCMV DNA homologous sequences were detected in 2 of 13 normal prostate, 2 of 9 BHP, and 3 of 10 ACP tissues, and HSV-2 DNA homologous sequences were found in 1 normal prostate tissue and 2 ACP tissues. In situ DNA-RNA hybridizations indicated HCMV-specific RNA in 3 of 8 BHP and 4 of 9 ACP tissues. No positive in situ hybridization for HCMV RNA was observed in normal prostate tissues. Parallel in situ DNA-RNA hybridization localized HSV-2-specific RNA in only 1 of 8 tumor sections. No HSV-2-specific RNA was observed in sections of normal and BHP tissues. Anticomplement immunofluorescence (ACIF) tests of BHP and ACP specimens showed specific HCMV immunofluorescence in 3 of 8 BHP and 4 of 9 ACP tissues. ACIF results correlate closely with in situ hybridization results and imply some degree of HCMV association with prostatic abnormality. The results also suggest that latent HCMV may be harbored by the human prostate gland.

Effect of 9-(2-hydroxyethoxymethyl)guanine on viral-specific polypeptide synthesis in human cytomegalovirus-infected cells.

Plaque-forming assay resulted in a 50 percent inhibitory dose by 9-(2-hydroxyethoxymethyl)guanine (acyclovir) against Towne strain human cytomegalovirus (HCMV) of approximately 98 mumol. At the drug concentration of 200 mumol, we did not detect any significant inhibition of viral DNA synthesis by cRNA-DNA hybridization. However, at this drug concentration, the synthesis of at least two viral-specific late polypeptides (150K and 67K) was significantly retarded up to 48 hours after infection, but resumed at 72 hours. These data suggest that acyclovir or its in vivo transformed derivative had a transient effect on viral-specific polypeptide synthesis in HCMV-infected human fibroblasts at a high drug concentration.

Antitumor agents 32. Synthesis and antitumor activity of cyclopentenone derivatives related to helenalin.

Several new cyclopentenones related to helenalin have been synthesized as potential alkylating antitumor agents. The procedure involved the transformation of 2-methyl-2-carbethoxycyclopentanone (2) to an ethylene ketal 3, bromination of 3 followed by dehydrobromination to yield a ketal olefin 5, reduction of 5 to the alcohol 6, conversion of 6 to the corresponding hydroxycyclopentenone 7, and estrification of 7 to afford the cyclopentenone esters 8--11. Biological assays indicated that only cyclopentenones possessing a conjugated ester side chain, such as 9 and 10, demonstrated significant in vitro cytotoxicity against the growth of tissue culture cells originating from human epidermoid carcinoma of the larynx (H.Ep.-2) as well as in vivo antitumor activity in Walker 256 carcinosarcoma in rats and P-388 lymphocytic leukemia in mice.

Antitumor agents. 21. A proposed mechanism for inhibition of cancer growth by tenulin and helenalin and related cyclopentenones.

Evidence is presented that sesquiterpene lactones or ketones containing the O=CC=CH2 moiety, e.g., tenulin and helenalin, alkylate the thiol group of reduced glutathione and L-cysteine in vitro. A proposal is offered that this mechanism of action is responsible for the observed potent in vivo antitumor activity of these agents in the Ehrlich ascites and Walker 256 carcinosarcoma and to a lesser extent in the P388 leukemic screen. Inhibition of tumor growth is thought to occur due to the O=CC=CH2 system alkylating by rapid Michael addition the SH biological nucleophiles of key regulatory enzymes of nucleic acid and chromatin metabolism. This proposition is in accord with the ability of these agents to inhibit DNA synthesis and gene activity of Ehrlich ascites cells.

Synthesis of isosteres of p-amidinophenylpyruvic acid. Inhibitors of trypsin thrombin, and pancreatic kallikrein.

A series of amino acids, amidino acids, and amidino esters was synthesized and the compounds were evaluated for their inhibitory activity against bovine trypsin, bovine thrombin, and porcine pancreatic kallikrein and as anticoagulants. Among these compounds, ethyl 4-amidino-2-iodophenoxyacetate was found to be the most effective inhibitor of the enzymes in question, with a potency (Ki = 3.16 x 10-6 M vs. trypsin; Ki = 4.8 x 10-5 M vs. thrombin) similar to that of p-amidinophenylpyruvic acid (Ki = 6.0 x 10-6 M vs. trypsin; Ki = 2.0 x 10-5 M vs. thrombin). Ethyl 4-amidino-2-iodophenoxyacetate was also found to be the most effective in blocking the clotting activity of plasma, as indicated by significant prolongation of the partial thromboplastin time. This paper reports the synthetic methods, the enzyme inhibitory activity, and the structure-activity relationships observed.

Some nucleoside analogs with anti-human immunodeficiency virus activity inhibit replication of Epstein-Barr virus.

The effects of (+)-beta-D-dioxolane-cytosine ((+)-D-beta-DOC), (-)-beta-L-dioxolane-cytosine ((-)-L-beta-DOC), (+)-beta-D-oxathiolane-cytosine ((+)-D-beta-OTC), (-)-beta-L-oxathiolane-cytosine ((-)-L-beta-OTC, or 3TC), 3'-azido-2',3'-dideoxy-5-methyl-cytidine (5-Me-AZDC), and 3'-azido-2',3'-dideoxyuridine (AZDU) on Epstein-Barr virus (EBV) DNA replication in vitro were tested in P3HR-1 cells. Two anti-EBV drugs, 3'-azido-3'-deoxythymidine (AZT) and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG, or ganciclovir), were used as positive controls. The inhibitory effects on EBV DNA synthesis were quantified by membrane filter and Southern blot hybridizations with an EBV-specific probe BamHI-W fragment. The 50% effective doses (ED50) for EBV DNA replication were 0.15, 0.83, 1.5, 8.3, 14, and 7.7 microM for DHPG, (-)-L-beta-DOC, (+)-D-beta-DOC, (+)-D-beta-OTC, (-)-L-beta-OTC, and AZT, respectively. In contrast, 5-Me-AZDC and AZDU were not effective at concentrations as high as 30 microM. These results indicated that both (-)-L-beta-DOC and (+)-D-beta-DOC were more potent than AZT, which has previously been shown to have anti-EBV activity. (-)-L-beta-DOC and (+)-D-beta-DOC have also been previously demonstrated to suppress the infectivity of human immunodeficiency virus type 1 (HIV-1). Thus, (-)-L-beta-DOC represents the first nucleoside analog with L-configuration exhibiting significant antiviral activities against both EBV and HIV.

Characterization of dynorphin-containing neurons on dissociated dentate gyrus cell cultures.

In the dentate gyrus, the synthesis of the opioid peptide, dynorphin, is modulated by a variety of stimuli. In order to elucidate the cellular and molecular mechanisms regulating the synthesis of dynorphin in the hippocampus, we have established a routine primary cell culture of dentate granule neurons and identified granule-like neurons by a characteristic marker, dynorphin, in these cultures. Cultures were prepared from 7-day-old rat pups and maintained in medium with 2% fetal bovine serum. These cultures contained approximately 20% neurons and survived for over 4 weeks. After 2 weeks in culture, neurons expressing dynorphin-A and its messenger RNA were detected using immunocytochemistry and in situ hybridization, respectively. In dentate cultures, enkephalin-, cholecystokinin-, neuropeptide Y- and substance P-positive cells were observed in addition to dynorphin-positive cells with immunocytochemistry. The results suggest that dentate gyrus cell cultures provide a valid in vitro model for studying molecular mechanisms regulating prodynorphin gene expression.

A strategy for precision of genotyping of Epstein-Barr virus by polymerase chain reaction: application for studying Hodgkin's lymphoma.

Previous studies on the genotyping of Epstein-Barr virus (EBV) have been based on the analysis of a single gene locus. The assignment of genotype of an isolate could easily be over-looked with this assay. Our strategy for precision of EBV genotyping has exploited the existence of two families of EBV strains (type A and B) that can be distinguished at three divergent gene loci (EBNA-2, EBNA-3C, and EBER). To precisely determine the genotype of EBV in Hodgkin's disease (HD), we designed primers and simultaneously analysed these three gene loci that distinguish type A and B viruses by the polymerase chain reaction (PCR) technique. The primers designed to amplify these three gene loci encompass either type-specific deletion sequences (EBNA-2 and EBNA-3C) or type-specific point mutations (EBER) that identify the virus strain based on the sizes of PCR-amplified products or the mobility shifts in single-strand conformation polymorphism (SSCP) analysis. The locations of point mutations were identified by direct sequencing of the PCR-amplified DNA. Fifteen EBV-infected cell lines were analysed and a good correlation between EBNA-2 and EBNA-3C typing results was found. In contrast, approximately 33% of the cell lines analysed maintained type A sequences in EBNA-2 and EBNA-3C genes while carrying type B sequences in the EBER region. Data obtained from analysis of cell lines served as a reference for studying HD samples. EBV DNA was detected in about 70% of HD. Among the EBV-positive samples, 56% were associated with type A virus, 13% with type B, and 31% with dual viral sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative study of herpes group virus-induced DNA polymerases.

A comparative biochemical study of virus-induced DNA polymerases was made among the herpes group viruses: namely, herpes simplex virus (HSV) type 1 and type 2, human cytomegalovirus (HCMV) and varicella-zoster virus (VZV). Although these virus-induced enzymes shared some biochemical properties, they differed in several important aspects. All these virus-induced DNA polymerases could efficiently use poly(dC) . oligo(dG)12--18 and poly(dA) . oligo(dT)12--18 as template-primers. However, in phosphocellulose chromatography, HSV-1- and HSV-2-induced enzymes were eluted at the low concentration of 0.18--0.20 M NaCl and the counterparts of HCMV and VZV were eluted at 0.30--0.32 M. The former two enzymes were more sensitive to lower concentrations of phosphonoacetate and ethyl phosphonoacetate than the latter two enzymes. Moreover, the activity of HSV-1- and HSV-2-specified DNA polymerases was 5 times greater in the presence of 60 mM ammonium sulfate if poly(dA) . oligo(dT)12--18 was used as template-primer, while HCMV- and VZV-induced enzyme activities were only about twice as great under the same conditions. Futhermore, DNase activity was conspicuous in both HSV-1- and HSV-2-infected WI-38 cells, but was not detectable in HCMV- and VZV-infected cells. After storage for 1 year at 4 degrees, the HSV-1-induced DNA polymerase was the most thermostable of the four viral enzymes.

Human cytomegalovirus-associated DNA polymerase and protein kinase activities.

Human cytomegalovirus (HCMV), purified exclusively from the extracellular media, contained a DNA polymerase activity in addition to a protein kinase activity. The DNA polymerase expressed its maximum activity in the presence of 5 to 10 mM-MgCl2. The enzyme was able to use effectively activated calf thymus DNA, poly(dA).oligo(dT)12--18 and poly(dC).oligo(dG)12--18 as the template primers. The DNA polymerizing activity was eluted with 0.18 to 0.2 M-KCl from a phosphocellulose column. It was relatively resistant to phosphonoacetic acid inhibition even at a high concentration of 100 micrograms/ml with activated calf thymus DNA as the template primer, but the DNA polymerase activity was totally suppressed at this concentration when poly(dA).oligo(dT)12--18 was used as the template primer. The enzyme activity was inhibited by ammonium sulphate at 0.01 to 0.3 M with either activated calf thymus DNA or poly(dA).oligo(dT)12--18 as the template primer. The protein kinase has maximum activity in the presence of 10 to 20 mM-MgCl2, and preferred virion proteins as phospho-acceptor to protamine sulphate. Histone, caesin and bovine serum albumin (BSA) were found to be poor substrates. The phosphorylated protein pattern of the in vivo [32P]orthophosphate-labelled virions was not identical to that of the in vitro phosphorylated Nonidet P40-dissociated virions, although seven phosphorylated polypeptides did co-migrate in SDS--polyacrylamide gel electrophoresis (SDS--PAGE). Procedures known to solubilize virions showed that the DNA polymerase and protein kinase were internal components of the virion.

Cytomegalovirus infection and viral-induced transformation of human endothelial cells.

Human cytomegalovirus (CMV) has been associated with vascular pathology. In vivo, CMV is present in vessel wall cells during acute and chronic infections as well as in atherosclerotic lesions. CMV nucleic acids and proteins have also been detected within Kaposi's sarcoma lesions. Because of these associations, we studied the interaction of CMV with human endothelial cells with particular attention to its oncogenicity in this cell type. Our data demonstrate that human endothelial cells are permissive to viral replication but that the viral replication cycle is delayed compared with fibroblast cells. Persistent infections can result with minimal cytopathology. CMV can transform these cells to anchorage-independent growth, and noninfectious virus is still capable of inducing this transforming event. Our results demonstrate that productive or persistent CMV infection of endothelial cells and viral-induced transformation can occur, thus providing an in vitro correlate of in vivo events.

Regulation of the expression of proenkephalin mRNA in bovine adrenal chromaffin cells: Role of proto-oncogenes.

The level of proenkephalin mRNA in bovine adrenal chromaffin cells was studied in the presence of cycloheximide, an inhibitor of translation, and two modulators of proenkephalin synthesis, nicotine and 12-O-tetradecanoylphorbol-13-acetate (TPA). Cycloheximide (CHX) abolished the induction of proenkephalin mRNA expression and protein synthesis by these two modulators, indicating that de novo protein synthesis was necessary for proenkephalin gene activation. The transcriptional regulatory regions of the proenkephalin gene displayed an extremely high degree of interspecies sequence conservation between humans, rats, and cows. In addition, molecular analyses of the human proenkephalin gene defined a cluster of responsive elements, designated as ENKCRE-1, ENKCRE-2, and AP-2; ENKCRE-2 acted functionally like both an AP-1 motif and a CAMP responsive element (CRE). When oligonucleotides containing ENKCRE-1, ENKCRE-2, AP-2, AP-1, and CRE motifs were used in protein-DNA gel mobility retardation experiments, the induction of ENKCRE-2/AP-1 activity correlated well with the level of proenkephalin mRNA induction. This ENKCRE-2/AP-1 complex could be inhibited by a specific c-Jun antiserum and was super-shifted by a polyclonal antibody against the Fos family of proteins. Furthermore, Western blot analysis suggested that c-Jun and Fos-related antigens rather than c-Fos per se were components of an ENKCRE-2/AP-1 complex. Thus, Fos-related proteins apparently form a complex with c-Jun that transactivates the proenkephalin gene.

Regulation of proenkephalin expression in C6 rat glioma cells.

The expression of proenkephalin (PENK) mRNA in C6 rat glioma cells was stimulated by norepinephrine (a beta-adrenergic agonist) and markedly enhanced by the addition of dexamethasone (a glucocorticoid agonist) to the culture medium, although dexamethasone alone exhibited no significant increase in PENK mRNA. Furthermore, no induction of glucocorticoid-response-element (GRE)-binding proteins was detectable. In contrast, the stimulation of PENK mRNA expression was not observed with a protein kinase C activator, 12-tetradecanoylphorbol-13-acetate (TPA), which stimulated the expression of c-fos and c-jun mRNA and their proto-oncoproteins (c-Fos and c-Jun). In addition, an AP-1 activity was induced by TPA and an induction of a kappaB-like binding activity was found with TPA plus cycloheximide-treated cells. Together, they suggest that activation of PENK gene in C6 cells is probably mediated mainly through the, beta-adrenergic agonist-elicited cyclic AMP signal pathway, and induction of AP-1 and kappaB-like binding activities appear not to participate in gene activation. Interestingly, the Western blot data showed no increase in intracellular levels of proenkephalin between control and treated cells. However, a marked increase in immunoreactivities for proenkephalin and its derivative, [Met(5)]-enkephalin was detected in medium and a lesser elevation in cells from modulator-treated cell culture through the time course. These results indicated that there was an association between an increase in PENK mRNA expression and an elevation of proenkephalins, and subsequently, the synthesized proenkephalins were released into the medium.

Purification and characterization of varicella-zoster virus-induced DNA polymerase.

Infection of WI-38 human fibroblasts with varicella-zoster virus led to the stimulation of host cell DNA polymerase synthesis and induction of a new virus-specific DNA polymerase. This virus-induced DNA polymerase was partially purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. This virus-induced enzyme could be distinguished from host cell enzyme by its chromatographic behavior, template specificity, and its requirement of salt for maximal activity. The enzyme could efficiently use poly(dC).oligo(dG)12-18 as well as poly(dA).oligo(dT)12-18 as template-primers. It required Mg2+ for maximal polymerization activity and was sensitive to phosphonoacetic acid, to which host alpha- and beta-DNA polymerase were relatively resistant. In addition, this induced DNA polymerase activity was enhanced by adding 60 mM (NH4)2SO4 to the reaction mixture.

Effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine on human cytomegalovirus replication in vitro.

We studied the effect of a novel purine acyclic nucleoside, 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), on human cytomegalovirus (HCMV) replication. The susceptibility of HCMV to this drug was monitored in cell culture by plaque reduction assay. HCMV replication of various strains was inhibited to the extent of 50% by 1 to 5 microM DHPG. DHPG was highly specific in its anti-HCMV activity, since at concentrations as high as 100 microM it did not exert any detectable inhibitory effect on uninfected cell macromolecular synthesis and cell growth. At concentrations of 2 to 4 microM, the drug inhibited the synthesis of six virus-specific polypeptides with molecular weights of 200,000 (VP200), 150,000 (VP150), 67,000 (VP67), 54,000 (VP54), 32,000 (VP32), and 27,000 (VP27) up to 96 h after infection. HCMV DNA synthesis was also considerably suppressed at concentrations of 2 to 4 microM DHPG. Upon removal of the inhibitor, however, viral DNA synthesis resumed and infectious virus reappeared, indicating that this inhibition was a virostatic reversible-type inhibition.

Functional characterization of partially purified Epstein-Barr virus DNA polymerase expressed in the baculovirus system.

The DNA polymerase gene of Epstein-Barr virus (EBV) was cloned into baculovirus transfer vector (pBlueBac). The recombinant baculovirus (AcEBP-15) was obtained by cotransfection of Spodoptera frugiperda (Sf9) cells with infectious DNA from Autographa californica multiple nuclear polyhedrin virus (AcMNPV) and pBlueBac plasmid carrying EBV polymerase gene. Infection of Sf9 cells with the recombinant virus produced substantial quantities of the EBV DNA polymerase protein of the expected size (110 kD). The identity of the EBV polymerase 110-kD polypeptide was determined by (a) immunoprecipitation and Western blot analyses with rabbit polyclonal antiserum specific for a synthetic peptide derived from the coding sequence of the polymerase gene; (b) identification of a polypeptide of identical size (110 kD) from EBV-infected cells; (c) measurement of DNA polymerase activity similar to that of the enzyme induced in EBV-infected cells; and (d) neutralization of the enzymatic activity by the rabbit antiserum and inhibition by phosphonoacetic acid. Our results indicate that the baculovirus expression system provides large quantities of functional polymerase suitable for biochemical and structural analyses, thereby furthering our understanding of the mechanism of viral DNA replication and its inhibition by antiviral drugs.

The Epstein-Barr virus BMLF1 promoter contains an enhancer element that is responsive to the BZLF1 and BRLF1 transactivators.

We have previously shown that the Epstein-Barr virus (EBV) immediate-early gene product, BZLF1, can activate expression of the EBV BMLF1 immediate-early promoter in EBV-positive, but not EBV-negative, B cells, suggesting that the BZLF1 effect may be mediated through another EBV gene product (S. Kenney, J. Kamine, E. Holley-Guthrie, J.-C. Lin, E.-C. Mar, and J. S. Pagano, J. Virol. 63:1729-1736, 1989). Here, we show that the EBV BRLF1 immediate-early gene product transactivates the BMLF1 promoter in either EBV-positive or EBV-negative B cells. Deletional analysis revealed that both the BZLF1-responsive region and the BRLF1-responsive region of the BMLF1 promoter are contained within the same 140-base-pair FokI-PvuII fragment located 300 base pairs upstream of the mRNA start site. This FokI-PvuII fragment functions as an enhancer element in the presence of the BRLF1 transactivator and contains the sequence CCGTGGAGA ATGTC, which is strikingly similar to the BRLF1-responsive region of the EBV DR/DL enhancer (A. Chevallier-Greco, H. Gruffat, E. Manet, A. Calender, and A. Sergeant, J. Virol. 63:615-623, 1989). The effect of BZLF1 on the BMLF1 promoter is likely to be indirect and mediated through the BRLF1 transactivator.

Inhibition of cellular DNA polymerase alpha and human cytomegalovirus-induced DNA polymerase by the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine.

The triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine were examined for their inhibitory effect on highly purified cellular DNA polymerase alpha and human cytomegalovirus (Towne strain)-induced DNA polymerase. These two nucleoside triphosphates competitively inhibited the incorporation of dGMP into DNA catalyzed by the DNA polymerases. The virus-induced DNA polymerase had greater binding affinity for the triphosphate of 9-(2-hydroxyethoxymethyl)guanine (Ki, 8 nM) than for the triphosphate of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (Ki, 22 nM), although the nucleoside of the latter compound was strikingly more effective against human cytomegalovirus replication in cell cultures than the nucleoside of the former. The Ki values of these two nucleoside triphosphates for alpha polymerase were 96 and 146 nM, respectively, and were 7- to 12-fold higher than those for the virus-induced enzyme. These data indicated that virus-induced DNA polymerase was more sensitive to inhibition by these two nucleoside triphosphates than was the cellular alpha enzyme.