Growth regulation of melanoma cells by interferon and (2'-5')oligoadenylate synthetase.

We report that endogenous, as well as exogenous, interferon (IFN) regulates the growth of human melanoma cells in culture. When antibodies directed against human fibroblast IFN were incorporated into the media of high-density cells stimulated to proliferate with serum, the cells entered the cell cycle earlier than did the controls. In investigating the biochemical basis for this finding, we have found that there is an inverse relationship between the (2'-5')oligoadenylate synthetase levels and the percentage of cells in S in untreated cultures. Upon IFN treatment, the relationship is obliterated and (2'-5')oligoadenylate synthetase levels increase throughout all phases of the cell cycle. This increase in enzyme levels correlates well with the decreased probability of the IFN-treated cells to cycle. These findings suggest a biological role for IFN as a negative growth factor for cells in culture.

Enhanced efficacy of the acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine in combination with gamma interferon against herpes simplex virus type 2 in mice.

The acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) and recombinant mouse interferon gamma (rMuIFN-gamma) were evaluated for their efficacy alone and in combination against a herpes simplex virus type 2 systemic infection in mice. Intraperitoneally infected animals were treated once a day with the drugs at various concentrations for 5 days starting 24 h after inoculation. DHPG was given subcutaneously and rMuIFN-gamma intraperitoneally. For DHPG, the effective dose at which 50% of the mice survived (ED50 was lowered approximately 10-fold from 3.4 to 0.25 mg/kg when given in combination with an ineffective dose of 4MuIFN-gamma (10(3) units per mouse). For rMuIFN-gamma, the ED50 was lowered greater than 10-fold from 6 x 10(3) to less than 3 x 10(2) units per mouse when given in combination with a marginally effective dose of DHPG (1 mg/kg). Construction of an isobologram and calculation of the corresponding fractional protective dose index (less than 0.12 where values less than or equal to 0.5 are considered synergistic) indicates an enhanced protective interaction by the combination of the two drugs.

Potent synergistic inhibition of herpes simplex virus-2 by 9-[(1, 3-dihydroxy-2-propoxy)methyl]guanine in combination with recombinant interferons.

DHPG, an acyclic guanine nucleoside with the structure 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine], showed potent synergism with recombinant alpha or beta interferons and modest synergism with gamma interferon in inhibiting the replication of herpes simplex virus type 2 in vitro. The most potent direct anti-herpes viral synergism was obtained by combination of DHPG and recombinant human interferon-beta-ser17; when combined, doses of each near their separate effective dose50's resulted in almost complete elimination of production of infectious virus within a single viral replication cycle. The anti-herpes viral activity of DHPG-interferon combinations was significantly greater than that obtained with acyclovir-interferon combinations.

Partial dissociation of antiviral and antimitogenic activities of murine interferon after its incorporation into liposomes.

Murine fibroblast interferon (MuIFN, 90% beta, 10% alpha) was associated with both positively and negatively charged liposomes formed by reverse-phase evaporation. This interferon-liposome association occurred predominantly in a manner that resulted in protection of a significant portion of the IFN's antiviral activity from trypsin digestion, yet also permitted biological expression of this activity without prior liposome disruption. A differential dissociation of antiviral and antimitogenic activities was observed with MuIFN associated with positively vs negatively charged liposomes, as reflected by differential sensitivities to trypsin inactivation. This may reflect either (1) differential associations of various molecular species of MuIFN with liposomes, or (2) that different portions of the IFN molecule are responsible for the antiviral and the antimitogenic activities.

Vaccinia virus and the EGF receptor: a portal for infectivity?

We previously demonstrated that occupancy of the epidermal growth factor (EGF) receptor reduced the ability of vaccinia virus to infect L cells [Eppstein et al: Nature 318:663, 1985]. This result suggested that vaccinia virus was utilizing the EGF receptor as one pathway to infect cells. We have studied this system further, and now find that antibodies to the EGF receptor also reduce the ability of vaccinia virus to infect cells productively. Inclusion of both EGF and antibodies to the EGF receptor did not cause inhibition over that obtained by EGF alone, providing another line of evidence that the antiviral effects on vaccinia virus were at the level of the EGF receptor. The antiviral effects of EGF or synthetic peptides corresponding to the third disulfide loop of TGF-alpha or the vaccinia virus growth factor were specific to vaccinia virus and did not inhibit replication of herpes simplex virus type 2 or vesicular stomatitis virus. The inhibitory effects on replication of vaccinia virus were obtained when EGF (but not insulin or growth hormone) was present prior to, but not after, productive viral adsorption. These results provided further evidence that the antivaccinia viral effects of EGF were at the level of initial receptor occupancy. As interferon (IFN) treatment has been shown to interfere with the action of some growth factors, including EGF, we examined the effects of IFN treatment of cells on the antivaccinia viral activity of EGF. Our results show that the antivaccinia effect of IFN-beta either interfered with or partially coalesced with the inhibitory effects of EGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of antiviral activity of (XyloA2'p)2XyloA.

(XyloA2'p)2xyloA, the xyloadenosine core analog of the interferon mediator 2-5A [ppp(A2'p)2A], was found to exhibit potent antiviral activity against herpes simplex viruses 1 and 2 (D. A. Eppstein, J. W. Barnett, Y. V. Marsh, G. Gosselin, and J. -L. Imbach (1983a) Nature (London) 302: 723-724). This xylo 2-5A core analog was over 100 times more stable to phosphodiester cleavage than was parent 2-5A core in cell-free extracts, which was originally thought to have contributed to its increased activity. However, we have now shown that the xylo 2-5A core does not activate the 2-5A-dependent endonuclease, and, additionally, that its mechanism of action is different from that of parent 2-5A core, even though it too does not activate the endonuclease. In fact, the mechanism of action of xylo 2-5A core apparently involves its degradation to monomer units, which then exert the antiviral effect. The enhanced antiviral activity of xylo 2-5A core (normalized to monomer-unit equivalents) as compared to that of xyloadenosine appears to be mediated through a slow release of xyloadenosine monomer units (i.e., xyloAMP). XyloAMP is resistant to inactivation by deamination, and thus intracellular xyloAMP should have increased antiviral activity compared to an equivalent concentration of xyloadenosine, which is subject to rapid inactivation by deamination.

Stereoconfiguration markedly affects the biochemical and biological properties of phosphorothioate analogs of 2-5A core, (A2'p5')2A.

The diester bonds of phosphorothioate trimer analogs of (A2'p5')2A (2-5A core) of the Sp stereoconfiguration were found to be extremely stable to hydrolysis by both serum and cellular phosphodiesterases. The corresponding Rp isomers, although still more stable than parent ppp(A2'p5')2A (2-5A), were significantly more susceptible to enzymatic hydrolysis than were the Sp isomers. Utilization of these novel 2-5A trimer isomers containing various combinations of Sp or Rp configurations at the internucleotidic phosphorothioate linkages revealed a further specificity of this enzymatic hydrolysis. Thus, the stereoconfiguration of the bond adjacent to the one undergoing hydrolysis influenced the rate of enzymatic hydrolysis, as well as did the chain length of the oligomer. The most stable trimer analog, which contained both internucleotide phosphorothioate linkages of the Sp configuration, had a half-life of 30 days in serum, which is a 1500-fold increase over that of parent 2-5A core. This is the first report on biochemical stability of an oligonucleotide containing more than one phosphorothioate linkage of the Sp configuration and is the first demonstration that a phosphorothioate internucleotide bond of the Sp configuration can increase the enzymatic stability of an adjacent phosphorothioate bond. In marked contrast to all previous 2-5A core analogs of increased stability, the activity (antiproliferative and antiviral) of the stable phosphorothioate 2-5A core analogs was obtained with the intact trimer, i.e., it was not attributed to antimetabolite degradation products.

Enhanced efficacy of the acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine in combination with beta-interferon against herpes simplex virus type 2 in mice.

The acyclic nucleoside 9-(1,3-dihydroxy-2- propoxymethyl )guanine (DHPG) and natural mouse interferon beta ( MuIFN -beta) were evaluated for their efficacy alone and in combination against herpes simplex virus type 2 systemic infections in mice. Intraperitoneally infected animals were treated once a day with the drugs at various concentrations for 5 days starting 24 h after inoculation. DHPG was injected subcutaneously at doses of 0.7 to 6 mg/kg. MuIFN -beta was given intraperitoneally at doses ranging from 3 X 10(3) to 3 X 10(4) IU per mouse. For DHPG alone, the effective dose at which 50% of the mice survived (ED50) was greater than 6 mg/kg. However, when given in combination with an ineffective dose of MuIFN -beta (10(4) IU per mouse), the ED50 for DHPG was 0.8 mg/kg. In addition, at the highest dose tested, MuIFN -beta alone had no protective activity against herpes simplex virus type 2 (ED50, greater than 3 X 10(4) IU per mouse). However, when given in combination with a marginally effective dose of DHPG (2 mg/kg), the ED50 for MuIFN -beta was less than 3 X 10(3) IU per mouse. Calculation of the fractional protective dose index (less than 0.23 where values of less than or equal to 0.5 are considered synergistic) indicates an enhanced protective interaction by the combination of the two drugs. These results represent the first time that potentiation of the antiviral activity of an acyclic nucleoside by interferon has been demonstrated in animal studies.

2-5A analogs as mechanistic probes of the 2-5A system: stereoconfiguration of phosphorothioate analogs of 2-5A markedly influences metabolic stability.

The stereoconfiguration of the diester bond of phosphorothioate analogs of 2-5A strongly influenced lability to enzymatic hydrolysis by cellular and serum phosphodiesterases. Bonds containing the Sp configuration were extremely resistant to hydrolysis compared to the corresponding Rp configuration linkages. The rate of hydrolysis of the diester bond was influenced by chain length of the adenylate oligomer, with a stability ranking of dimer greater than trimer greater than tetramer; as well as by the stereo-configuration of the diester bond adjacent to the one undergoing hydrolysis. The anti-proliferative and anti-viral activities of these various phosphorothioate 2-5A core analogs were assessed. The most stable analogs possessed the greatest biological activities (at 25-50 microM), which were not readily attributable to 2-5A degradation products or endonuclease activation. A 5'-triphosphate analog of 2-5A containing a phosphorothioate substituent in the gamma-position was obtained in good yield by enzymatic synthesis from ATP-gamma S. This gamma-thio 2-5A analog showed full biological activity.

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide.

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.

Enhanced efficacy of the acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine in combination with alpha-interferon against herpes simplex virus type 2 in mice.

The acyclic nucleoside DHPG [9-(1,3-dihydroxy-2-propoxymethyl)guanine] and recombinant human alpha-interferon of clones A/D potentiate each other's antiviral activity against a systemic infection with herpes simplex virus type 2. The effective dose at which 50% of the mice survived was lowered approximately 10-fold for DHPG when it was given in combination with a marginally effective dose of alpha-interferon and greater than 10-fold for alpha-interferon when it was given in combination with a nontherapeutic dose of DHPG.

Xyloadenosine analogue of (A2'p)2A inhibits replication of herpes simplex viruses 1 and 2.

Molecules of the structure ppp(A2'p)2A containing a 2' leads to 5' phosphodiester bond, commonly abbreviated as 2-5A, are synthesized in interferon-treated virally-infected cells and have been implicated in several systems as contributing to interferon's antiviral activity. The 2-5A binds to and subsequently activates an endogenous endonuclease, ultimately resulting in degradation of RNA. We have been interested in the use of 2-5A analogues to achieve antiviral activity without the use of interferon. For this approach to be successful, analogues must be synthesized with an increased stability (native 2-5A is rapidly degraded by cellular phosphodiesterases) and with increased ability to enter intact cells. Removal of the highly-negative charged 5' terminal phosphates from ppp(A2'p)2A results in formation of the 'core' species, (A2'p)2A, which should be able to penetrate intact cells more readily. While Kimchi et al. have shown that 2-5A core has an antimitogenic effect in mouse spleen lymphocytes and 3T3 fibroblasts, Williams and Kerr have reported lack of antiviral activity against Semliki Forest virus or encephalomyocarditis virus by exogenously-administered 2-5A core. We have previously determined that (xyloA2'p)2xyloA (abbreviated as xylo 2-5A core), the xyloadenosine analogue of the 5'-terminally dephosphorylated 2-5A core, is over 100 times more stable than the parent 2-5A core species. We now report that this xylo 2-5A core inhibits replication of herpes simplex viruses 1 and 2 in vitro, with greater than 100 times the activity of the parent 2-5A core. The mechanism of antiviral action of the 2-5A core analogue appears to involve a pathway different from that activated by the parent 5' triphosphorylated 2-5A species.

Enhanced antitumor efficacy of adriamycin in combination with interferon-gamma: effects on macrophages.

The combination of the immunomodulator interferon-gamma (IFN-gamma) with the chemotherapeutic drug adriamycin (ADM) was assessed in vitro and in vivo in murine tumor models. When tested in vivo against the murine Lewis lung carcinoma, significantly greater reduction of spontaneous pulmonary metastases was obtained by combination treatment with IFN-gamma, followed 1 day later by ADM. Intraperitoneal ADM treatment also resulted in an increased recruitment of peritoneal mononuclear cells. It is noteworthy that, although the antitumor efficacy was significantly increased by the IFN-gamma/ADM combination treatment, gross toxicity of ADM was not increased. Thus, a net increase in the therapeutic index of ADM was achieved. In vitro, the effects of ADM on the ability of murine peritoneal macrophages, with or without the addition of immunological macrophage activators, to kill tumor cells was studied. Resident macrophages were able to sequester ADM (when present at 10 micrograms/ml) from the medium, and could subsequently mediate killing of target tumor cells. However, incubation of macrophages with low (ineffective by themselves) doses of ADM (1 microgram/ml) prevented their simultaneous or subsequent activation to the tumoricidal state after incubation with the normal macrophage-activating mixture of IFN-gamma plus a muramyl dipeptide (MDP) analog. When the order of addition of reagents was reversed such that the macrophages were preincubated for 24 hr with IFN-gamma (100 U/ml) plus the MDP analog (0.1-10 micrograms/ml), no antagonism of tumoricidal activity was obtained upon subsequent incubation with ADM. There were no interactions between IFN-gamma and ADM on the direct proliferation of tumor cells. Taken together, these results suggest that the enhanced antitumor efficacy of IFN-gamma/ADM combinations in vivo was not due to direct antiproliferative effects on the tumor cells, but rather may be mediated by direct cytotoxicity of ADM on tumor cells enhanced by phagocytic mononuclear cells.

Biological activity of liposome-encapsulated murine interferon gamma is mediated by a cell membrane receptor.

Recombinant murine gamma interferon (rMuIFN-gamma) was found to bind reversibly to a specific high-affinity surface receptor on L929 cells; neither murine alpha or beta nor human gamma IFN competed for receptor binding. Encapsulation of the rMuIFN-gamma in either negatively or positively charged liposomes reduced its immediate ability to bind to this surface receptor. Disruption of liposome integrity with detergent resulted in full ability of the rMuIFN-gamma to bind to the membrane receptor. Incubation of the liposomal IFN in serum-containing medium resulted in significant leakage so that the IFN was able to bind to its surface receptor. Assessment of the biological activity of the rMuIFN-gamma preparations revealed that full antiviral activity was observed in vitro with the liposomal IFN preparations without their prior disruption by detergent. The antiviral activity observed with either free or liposomal IFN was neutralized completely by antibodies against rMuIFN-gamma. Both free and liposomal rMuIFN-gamma, in conjunction with bacterial lipopolysaccharide, were also able to activate murine peritoneal macrophages to the tumoricidal state. Again, this activity of both free and liposomal IFN could be neutralized completely by antibody. These results indicate that although rMuIFN-gamma can be effectively incorporated into liposomes, it must ultimately leak out of the liposome in order to mediate its biological effects; these effects are triggered after the IFN binds to its cell surface receptors.

Epidermal growth factor receptor occupancy inhibits vaccinia virus infection.

Vaccinia virus encodes VGF, an early protein of relative molecular mass 19,000 (19K) which, from amino-acid residues 45 to 85, is homologous in 19 residues to epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha). The conserved sequence includes a region of high homology (6 out of 10 amino acids) from residues 71 to 80, corresponding to the third disulphide loop of both EGF and TGF-alpha. This region has recently been shown to contain a binding region of TGF-alpha for the EGF receptor, and this raises the question of whether vaccinia virus utilizes the EGF receptor in order to bind to and infect cells. We now show that occupancy of the EGF receptor inhibits vaccinia virus infection. Inhibition is observed in a dose-dependent fashion by pre-treatment with either EGF or synthetic decapeptide antagonists of EGF's mitogenic activity which correspond to the sequence of the third disulphide loop of VGF or TGF-alpha. The relative ability of the peptides to inhibit vaccinia virus infection parallels their binding affinity to the EGF receptor.

Inhibition of vesicular stomatitis virus replication in dexamethasone-treated L929 cells.

We previously demonstrated that dexamethasone treatment of L929 cells inhibited plaque formation by vesicular stomatitis virus (VSV), encephalomyocarditis virus, or vaccinia virus. We now have characterized the antiviral effects of glucocorticoids in L929 cells. Dexamethasone did not directly inactivate VSV nor did steroid treatment of L929 cells affect virion adsorption or penetration. The VSV yield in L929 cells treated with dexamethasone for a period of only 4 or 8 hr was decreased by 50% when cells were infected the day following steroid treatment. Treating L929 cells with dexamethasone for a longer period resulted in greater inhibitions of virus synthesis. Interferon activity (less than 5 units/ml) was not detected in L929 cell culture fluids and cell sonicates from steroid-treated cells and the addition of antiserum to murine alpha/beta-interferon had no effect on the ability of dexamethasone to inhibit VSV replication. Dexamethasone treatment of L929 cells did not induce the production of double-stranded RNA-dependent protein kinase but did result in a slight elevation of 2-5A oligoadenylate synthetase activity, two enzymatic activities associated with the antiviral state induced by interferon. However, the elevated 2-5A synthetase activity was not associated with an inhibition of VSV RNA accumulation in dexamethasone-treated L929 cells. By contrast, the synthesis of all five VSV proteins was reduced by 50-75% in dexamethasone-treated L929 cells as early as 4 hr after infection. Thus, the dexamethasone-mediated inhibition of VSV replication in L929 cells is associated with decreased production of VSV structural proteins.