Self-tolerance to the murine homologue of a tyrosinase-derived melanoma antigen: implications for tumor immunotherapy.

The human tyrosinase-derived peptide YMDGTMSQV is presented on the surface of human histocompatibility leukocyte antigen (HLA)-A*0201(+) melanomas and has been suggested to be a tumor antigen despite the fact that tyrosinase is also expressed in melanocytes. To gain information about immunoreactivity and self-tolerance to this antigen, we established a model using the murine tyrosinase-derived homologue of this peptide FMDGTMSQV, together with transgenic mice expressing the HLA-A*0201 recombinant molecule AAD. The murine peptide was processed and presented by AAD similarly to its human counterpart. After immunization with recombinant vaccinia virus encoding murine tyrosinase, we detected a robust AAD-restricted cytotoxic T lymphocyte (CTL) response to FMDGTMSQV in AAD transgenic mice in which the entire tyrosinase gene had been deleted by a radiation-induced mutation. A residual response was observed in the AAD(+)tyrosinase(+) mice after activation under certain conditions. At least some of these residual CTLs in AAD(+)tyrosinase(+) mice were of high avidity and induced vitiligo upon adoptive transfer into AAD(+)tyrosinase(+) hosts. Collectively, these data suggest that FMDGTMSQV is naturally processed and presented in vivo, and that this presentation leads to substantial but incomplete self-tolerance. The relevance of this model to an understanding of the human immune response to tyrosinase is discussed.

A putative role for nicotinamide adenine dinucleotide-promoted nuclear protein modification in the antitumor activity of N-methyl-N-nitrosourea.

Incubation of HeLa cells with the anticancer agent N-methyl-N-nitrosourea (MNU) results in: (a) depression of intracellular nicotinamide adenine dinucleotide levels; (b) stimulation of the chromatin-associated, chromosomal protein-modifying enzyme polyadenosine diphosphoribose [poly(ADP-ribose)] polymerase, which uses nicotinamide adenine dinucleotide as substrate; and (c) some fragmentation of cellular DNA. DNase treatment of HeLa nuclei in vitro also stimulates poly(ADP-ribose) polymerase activity, but not in nuclei derived from MNU-treated cells unless they have been subsequently incubated to allow for recovery from MNU damage. DNA polymerase activity is stimulated in vitro by poly(ADP) ribosylation of nuclear proteins. By using intact nuclei derived from MNU-treated HeLa cells, the repair via elongation of single-strand DNA breaks is demonstrated in vitro. This repair is dependent on DNA polymerase activity and is enhanced by adenosine diphosphate ribosylation of histones. Inhibition of poly(ADP-ribose) polymerase with nicotinamide results in extensive degradation of MNU-damaged DNA. Taken as a whole, these results suggest that poly(ADP-ribose) polymerase may play a role in the repair of alkylation damage to cellular DNA and that the inhibition of this enzyme in vivo might be exploited to potentiate the antitumor and carcinogenic activities of MNU.

Tumor-induced immune dysfunction: the macrophage connection.

Although macrophages (Mphis) mediate tumor cytotoxicity, display tumor-associated antigens, and stimulate antitumor lymphocytes, cancer cells routinely circumvent these host-mediated immune activities, rendering the host incapable of mounting a successful antitumor immune response. Evidence supporting a direct causal relationship between cancer and immune dysfunction suggests that the presence of neoplastic tissue leads to immunologic degeneration. Furthermore, substantial data demonstrate that tumor growth adversely alters Mphi function and phenotype. Thus, although Mphis can serve as both positive and negative mediators of the immune system, the importance of Mphis in tumor-induced immune suppression remains controversial. This review focuses on the evidence that tumor-derived molecules redirect Mphi activities to promote tumor development. Tumors produce cytokines, growth factors, chemotactic molecules, and proteases that influence Mphi functions. Many tumor-derived molecules, such as IL-4, IL-6, IL-10, MDF, TGF-beta1, PGE2, and M-CSF, deactivate or suppress the cytotoxic activity of activated Mphis. Evidence that tumor-derived molecules modulate Mphi cytotoxicity and induce Mphi suppressor activity is presented. This information further suggests that Mphis in different in vivo compartments may be differentially regulated by tumor-derived molecules, which may deactivate tumor-proximal (in situ) Mphi populations while concurrently activating tumor-distal Mphis, imparting a twofold insult to the host's antitumor immune response.

Tumor cell-derived TGF-beta and IL-10 dysregulate paclitaxel-induced macrophage activation.

Paclitaxel (TAXOL) activates in vitro macrophage (Mø) expression of proinflammatory and cytotoxic mediators, including IL-12, tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO). However, tumors dysregulate Mø through soluble suppressor molecules, and it is possible that tumors evade paclitaxel-mediated immune effector function through the production of immunomodulatory molecules and inhibition of Mø function in situ. Because Mø activation in the tumor microenviroment is a desirable goal of anti-tumor immunotherapy, we evaluated whether tumor-derived immunomodulatory factors dysregulate paclitaxel-mediated Mø activation. Tumor cell-derived supernatant suppressed paclitaxel's capacity to induce IL-12, TNF-alpha, and NO production by RAW264.7 Mø. Tumor factors also dysregulated paclitaxel-induced expression of a HIV-LTR, promoter-driven luciferase construct in RAW264.7 Mø, suggesting that tumors may inhibit a broad range of Mø functionality. Depletion studies revealed that IL-10 and transforming growth factor-beta1 (TGF-beta1), but not prostaglandin E2 (PGE2), impaired paclitaxel-mediated activation, suggesting that abrogation of these factors in situ might restore paclitaxel's activating capacity and enhance anti-tumor efficacy.

Effect of hyperthermia and doxorubicin on nucleoid sedimentation and poly (ADP-ribose) polymerase activity in L1210 cells.

We report on the individual and combined effects of doxorubicin (DOX) and hyperthermia (HYP) on nucleoid sedimentation and poly (ADP-ribose) polymerase (PARP) activity of L1210 cells. The effects of HYP and DOX on nucleoid sedimentation (increased sedimentation) were similar and correlated with cell viability. No correlation of PARP activity with cell toxicity was evident; the activity of PARP was inhibited by HYP (42 degrees C; 1-3 h) and stimulated by DOX (1-10 microM; 30 min). The HYP-induced inhibition of PARP was actually ameliorated by simultaneous exposure to DOX. Although separate studies have previously suggested that chromatin alterations or the inhibition of PARP might play a role in the effect of HYP, the correlation of nucleoid changes (rather than PARP activity) with cell viability emphasizes the contribution of the former. Furthermore, the results suggest that the nucleoid technique may prove useful in screening potential treatment modalities.

Aliphatic amino acid side chains associate with the "face" of the adenine ring.

We have synthesized the free amino acid adenylate anhydrides of phenylalanine, leucine, isoleucine and valine. These activated compounds are very labile at high pH, but at low pH they become more stable. Proton NMR spectra of these adenylates show that in every case, the hydrophobic side chains, even in these small molecules at low pH and low concentration, are associated with the "face" of the adenine ring. Although aromatic rings are known to associate with adenine in this fashion, to our knowledge this is the first report of an intercalative-type interaction of aliphatic side chains with nucleic acid bases. Since adenine is the most hydrophobic base, these interactions are of a hydrophobic character, and occur in spite of the fact that the adenine ring is protonated. These results may have implications regarding recognition processes in DNA-protein and RNA-protein interactions.

The science literacy crisis, philosophical issues, and the origin sciences.

Since 1983, when the National Commission on Excellence in Education released its much-awaited report, A Nation at Risk, the United States has perceived itself to be in the midst of a serious education crisis. While nearly everyone is in agreement that most areas of the educational enterprise are in need of some revision and revitalization, problems related to science education have clearly received the lion's share of reform-minded attention. The present essay reviews the various symptoms of the so-called science literacy (education) crisis; speculates on the unique role that the philosophical underpinnings of the "cosmic" (Weisskopf, 1994) or origin sciences may play in these problems; and offers some suggestions as to how academic research scientists interested in these areas might more readily and effectively involve themselves in efforts to improve science education at all levels of the educational system.

Studies of the chemical basis of the origin of protein synthesis: initiation and direction of peptide growth.

The data presented in this paper show that the ease of non-enzymatic activation of carboxylic acids by ATP at pH 5 varies directly with the pKa of the carboxyl group, and is consistent with the idea that it is the protonated form of the carboxyl group which participates in the activation reaction. Consequently, since most N-blocked amino acids have higher pKa's than do their unblocked forms, they are activated more readily, and we have demonstrated that this principle applies to peptides as well, which are activated more rapidly than single amino acids. We propose that this fact may be partly responsible for the origin of two important features still observed in contemporary protein synthesis: (1) initiation in prokaryotes is accomplished with an N-blocked amino acid, and (2) elongation in all living systems occurs at the carboxyl end of the growing peptide.

Comparative rates of esterification of 5'-AMP with hydrophobic amino acids: relevance to the genetic-code assignments.

We have continued our program aimed at understanding the origin and evolution of the genetic code and the process of protein synthesis by comparing the rates of esterification of 5'-AMP by a series of hydrophobic N-acetylamino acids. The reaction clearly shows differences in reaction rate (AcPhe greater than AcLeu greater than AcVal greater than AcIle) among the amino acids having A as middle letter of their anticodons. However, there were no significant differences in reaction rate between AcLeu, AcNorleu, and Ac-alpha-aminobutyric acid, and AcGly reacted faster than all of these and AcPhe. Consequently, this simple reaction with AMP can distinguish only among those amino acids that actually have A as the middle anticodonic nucleotide. The relevance of these studies to the origins of the process of protein synthesis and of the genetic code is discussed in conjunction with results from other studies of a similar nature.

Tumor growth modulates macrophage nitric oxide production following paclitaxel administration.

The antineoplastic agent paclitaxel (Taxol) mimics bacterial lipopolysaccharide (LPS) in normal host macrophages (Mphis), enhancing antitumor cytotoxicity in vitro. Because paclitaxel is used as an antitumor chemotherapeutic agent and tumor growth alters Mphi phenotype and function, we assessed effector molecule production and cytotoxic activity by normal host and tumor-bearing host (TBH) Mphis following paclitaxel administration. Paclitaxel treatment, duplicating human chemotherapeutic regimens, primed normal host splenic Mphis for enhanced production of the cytotoxic mediator nitric oxide (NO); in contrast, paclitaxel's NO-inducing activity was significantly suppressed in TBHs. In contrast to NO regulation, Mphi capacity for tumor necrosis factor-alpha (TNF-alpha) production in both normal hosts and TBHs was enhanced by paclitaxel administration. Although tumor growth modulated paclitaxel-induced Mphi NO production, paclitaxel administration enhanced both normal host and TBH Mphi cytotoxic antitumor activity. Blocking NO with a competitive inhibitor abrogated Mphi cytotoxicity, suggesting paclitaxel-induced TBH Mphi NO production, although suboptimal, remains sufficient to mediate antitumor activity. These data demonstrate that paclitaxel's in vivo immune activities are differentially regulated during tumor burden and suggest that paclitaxel's immunotherapeutic functions may contribute to its success as an anticancer agent.

Paclitaxel enhances macrophage IL-12 production in tumor-bearing hosts through nitric oxide.

Tumor-induced macrophages (Mphis) mediate immunosuppression, in part, through increased production of factors that suppress T cell responsiveness and underproduction of positive regulatory cytokines. Pretreatment of tumor-bearing host (TBH) Mphis with the anticancer agent paclitaxel (Taxol) partially reverses tumor-induced Mphi suppressor activity, suggesting that paclitaxel may restore TBH Mphi production of proimmune factors. Because paclitaxel demonstrates LPS-mimetic capabilities and increased production of the LPS-induced immunostimulatory cytokine IL-12 could account for enhanced T cell responsiveness, we investigated whether paclitaxel induces Mphi IL-12 production. Tumor growth significantly down-regulated Mphi IL-12 p70 production through selective dysregulation of IL-12 p40 expression. LPS stimulation failed to overcome tumor-induced dysregulation of p40 expression. In contrast, paclitaxel significantly enhanced both normal host and TBH Mphi IL-12 p70 production in vitro, although TBH Mphi IL-12 production was lower than that of similarly treated normal host Mphis. Paclitaxel enhanced p40 expression in a dose-dependent manner. Through reconstituted Mphi IL-12 expression, paclitaxel pretreatment relieved tumor-induced Mphi suppression of T cell alloreactivity. Blocking Mphi NO suppressed paclitaxel's ability to induce IL-12 production. This suggests that paclitaxel-induced activities may involve a NO-mediated autocrine induction pathway. Collectively, these data demonstrate that paclitaxel restores IL-12 production in the TBH and ascribe a novel immunotherapeutic component to the pleiotropic activities of NO. Through its capacity to induce IL-12 production, paclitaxel may contribute to the correction of tumor-induced immune dysfunction.

Rationalization of some genetic anticodonic assignments.

The genetic code appears to be a logic matrix in which, generally speaking, there is a correlation between the hydrophobicities of amino acids and their anticodonic nucleotides. There are several exceptions to this generality, however, and using previous data on hydrophobicity and binding constants, coupled with new data on reaction rates, we rationalize several of the anticodonic assignments.

Poly(adenosine diphosphate-ribose) polymerase: the distribution of a chromosome-associated enzyme within the chromatin substructure.

The distribution of a chromatin-bound, nuclear protein modifying enzyme, poly (adenosine diphosphate-ribose) polymerase, and its product, poly(ADP-ribose), among various fractions of sheared and nuclease-digested HeLa cell chromatin has been examined. Epichlorohydrin-tris(hydroxymethyl)aminomethane-cellulose and glycerol gradient fractionation of solubilized chromatin indicated that poly(ADP-ribose)polymerase activity was associated primarily with the template active regions (euchromatin), whereas the transcriptionally inert chromatin fractions were found to contain relatively low levels of ADP-ribosylating activity. When isolated HeLa cell nuclei were digested in situ with micrococcal nuclease and the resultant chromatin was fractionated into nucleosome monomers (v bodies) and oligomers by sucrose gradient centrifugation, only material sedimenting faster than the 11S monomers was found to contain appreciable poly(ADP-ribose) polymerase activity. If, on the other hand, isolated HeLa cell nuclei were first incubated with labeled NAD, the substrate for poly(ADP-ribose) polymerase, prior to the preparation and fractionation of nuclease-digested chromatin, it was found that those chromatin fractions which possess significant poly(ADP-ribose) polymerase activity (nucleosome oligomers) are relatively deficient in the labeled product of this enzyme, and that a considerable portion of the homopolymeric product is ultimately associated with the 11S v bodies. Additional evidence is presented which indicates that the absence of nucleosome monomer-associated poly(ADP-ribose) polymerase activity is not due to the absence of a suitable acceptor on these structures, and that the activity of this enzyme within the chromatin is most probably dependent upon the physical integrity of the oligomeric structures themselves.

The case for the anticode.

The present paper will focus on the developments in our lab related to the origin of the code since the Israel meeting (1,2). Principally these items are: (a) a new set of correlations (3) which include ranked hydrophobicities of amino acids and dinucleotides; (b) binding constants (4) of Phe for the four mononucleotides; and (c) binding constants (5) of Phe, Leu, Ile, Val, and Gly for polyadenylic acid (poly A). The data continue to support a model for the origin of the code based on relationships between amino acids and their anticodons.

Aminoacyl-nucleotide reactions: studies related to the origin of the genetic code and protein synthesis.

In the present paper, we report on the effect of pH and carbonate on the hydrolysis rate constants of N-blocked and free aminoacyl adenylate anhydrides. Whereas the hydrolysis of free aminoacyl adenylates seems principally catalyzed by OH-, the hydrolysis of the N-blocked species is also catalyzed by H+, giving this compound a U-shaped hydrolysis vs. pH curve. Furthermore, at pH's less than 8, carbonate has an extreme catalytic effect on the hydrolysis of free aminoacyl-AMP anhydride, but essentially no effect on the hydrolysis of N-blocked aminoacyl-AMP anhydride. Furthermore, the N-blocked aminoacyl-AMP anhydride is a very efficient generator of peptides using free glycine as acceptor. The possible significance of the observations to prebiological peptide synthesis is discussed.

Binding constants of phenylalanine for the four mononucleotides.

Earlier work has shown that several properties of amino acids correlate directly with properties of their anticodonic nucleotides. Furthermore, in precipitation studies with thermal proteinoids and homopolyribonucleotides, an anticodonic preference was displayed between Lys-rich, Pro-rich and Gly-rich thermal proteinoids and their anticodonic polyribonucleotides. However, Phe-rich thermal proteinoid displayed a preference for its codonic nucleotide, poly U. This inconsistency seemed to be explained by a folding in of the hydrophobic residues of Phe causing the proteinoid to appear more hydrophilic. The present work used nuclear magnetic resonance techniques to resolve a limited question: To which of the four nucleotides does Phe bind most strongly? The results show quite clearly that Phe binds most strongly to its anticodonic nucleotide, AMP.

Hydrolytic properties of phenylalanyl- and N-acetylphenylalanyl adenylate anhydrides.

We have used a novel spectrophotometric method to study the hydrolysis of N-acetylphenylalanyl adenylate anhydride (AcPhe-AMP) and phenylalanyl-adenylate anhydride (Phe-AMP) at low concentrations (10(-5) M), 25 degrees C, constant buffer concentration (0.05 M), and as a function of pH. While Phe-AMP is susceptible principally to attack by OH-, with two different rates depending on whether the alpha-amino group of the amino acid is protonated or not, the AcPhe-AMP is susceptible to acid decomposition as well. At pH's 4-8, the Phe-AMP hydrolyzes faster than the AcPhe-AMP, but at pH less than 4 or pH greater than 8, the blocked form hydrolyzes faster. Both forms are also attacked by H2O, and at the same rate. Moreover, the hydrolysis of Phe-AMP is shown to be greatly catalyzed by carbonate, although the AcPhe-AMP is not subject to such catalysis. The rate laws for the various mechanisms and the activation energies for the hydrolyses at pH 7.1 are given.

Taxol, a microtubule-stabilizing antineoplastic agent, differentially regulates normal and tumor-bearing host macrophage nitric oxide production.

Taxol, a potent antitumor chemotherapeutic, promotes in vitro cytotoxic antitumor activities by normal host macrophage (M phi s). Because tumor growth induces functional changes among M phi populations, we determined whether fibrosarcoma growth (Meth-KDE) modified M phi responsiveness to the activating agent taxol. Tumors induce tumor-distal M phi populations to become immune suppressor cells, partially through overproduction of the cytotoxic and proinflammatory molecules nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha). Beneficial to the tumor-bearing host (TBH) when released by tumor-proximal M phi s, NO and TNF-alpha suppress lymphoproliferation and fail to impart antitumor activity when expressed in tumor-distal compartments. We report that taxol differentially regulated normal host and TBH M phi production of the immunosuppressive molecule NO by tumor-distal M phi populations. In response to IFN-gamma-priming and taxol triggering, TBH M phi s increase their production of NO as compared to resting M phi s; however, unlike normal host M phi s, taxol-induced TBH M phi NO production was significantly suboptimal. Modulation of TBH M phi NO production in tumor-distal compartments may alleviate M phi-mediated suppression of T-cell proliferative responses, yet promote sufficient NO production by tumor-associated M phi s to affect cytotoxicity. Collectively, these data leave implications for immunotherapeutic activities by the anticancer drug taxol.

Interleukin-12 overcomes paclitaxel-mediated suppression of T-cell proliferation.

The antineoplastic agent paclitaxel (TAXOL) is a potent inhibitor of tumor cell division and a useful chemotherapeutic for the treatment of refractory ovarian and breast carcinoma. Multiple immune system actions have been ascribed to paclitaxel, including the capacity to induce macrophage antitumor cytotoxic molecule production. However, T-cells are susceptible to paclitaxel's cytostatic functions, and no studies have investigated the effects of direct paclitaxel administration on lymphocyte function in the tumor-bearing host (TBH). Because paclitaxel is currently used as an antitumor chemotherapeutic agent and tumor growth alters leukocyte functions, we assessed T-cell function following chemotherapeutic-type paclitaxel treatment. Paclitaxel administration significantly compromised the proliferative capacity of both normal host and TBH lymphocytes in vitro. Although tumor growth impaired T-cell interferon-gamma (IFN-gamma) production, paclitaxel treatment did not alter IFN-gamma. We speculate that the immunostimulatory cytokine interleukin-12 (IL-12), which promoted T-cell activation and proliferation, was capable of reversing paclitaxel-mediated immunosuppression. Exogenous IL-12 fully reconstituted proliferative reactivity and enhanced IFN-gamma production by both normal host and TBH lymphocytes in vitro. Collectively, these data suggest that chemotherapeutic paclitaxel regimens impart significant but reversible inhibition of lymphocyte populations, and IL-12 may be a useful ancillary immunotherapeutic to overcome paclitaxel-induced modulation of lymphocyte activities.

Manipulation of avidity to improve effectiveness of adoptively transferred CD8(+) T cells for melanoma immunotherapy in human MHC class I-transgenic mice.

The adoptive transfer of tumor-reactive CD8(+) T cells into tumor-bearing hosts provides an attractive alternative to vaccination-based active immunotherapy of melanoma. The development of techniques that result in the preferential expansion of tumor-reactive T cells is therefore of great importance. In this study, we report the generation of HLA-A*0201-restricted CD8(+) T cell populations that recognize either tyrosinase(369-376) or gp100(209-217) from tolerant human class I MHC-transgenic mice by using single amino acid-substituted variant peptides. Low peptide concentration or restimulation with the parent peptide was used to enhance the functional avidity, defined by stimulation of IFN-gamma accumulation, and cross-reactivity of the resulting T cell populations. We found a direct correlation between the ability of a T cell population to respond in vitro to low concentrations of the precise peptide expressed on the tumor and its ability to delay the outgrowth of B16 melanoma after adoptive transfer. Surprisingly, we found that some T cells that exhibited high functional avidity and were effective in controlling tumor outgrowth exhibited low structural avidity, as judged by MHC-tetramer staining. Our results establish strategies for the development and selection of CD8(+) T cell populations that persist despite peripheral tolerance, and that can control melanoma outgrowth. Furthermore, they support the use of human MHC class I-transgenic mice as a preclinical model for developing effective immunotherapies that can be rapidly extended into therapeutic settings.