Cell-mediated immune response to syngeneic UV induced tumors: I. The presence of tumor associated macrophages and their possible role in the in Vitro generation of cytotoxic lymphocytes

A primary in vitro sensitization system employing a chromium release assay was utilized to investigate reactivity of murine spleen cells toward syngeneic ultraviolet (uv) light induced fibrosarcomas. These tumors are immunologically rejected in vivo when implanted into normal syngeneic mice but grow progressivly when implanted into syngeneic mice that had previously been irradiated with subcarcinogenic levels of uv light. Following appropriate sensitization, spleen cells from both normal and uv irradiated mice are capable of developing cytotoxic lymphocytes in vitro against the uv induced tumors. It was subsequently discovered that in situ uv induced tumors all contained macrophages of host origin that became demonstrable only after enzymatic dissociation of the tumor tissue. These macrophages were immunologically active in vitro as their presence in the stimulator cell population was necessary to achieve an optimum anti-tumor cytotoxic response following in vitro sensitization. Anti-tumor reactivity generated by mixing spleen cells and tumor cells in the absence of tumor derived macrophages could be greatly enhanced by the addition of normal syngeneic peritoneal macrophages. When in vitro anti-tumor reactivity of spleen cells from normal and uv treated mice was compared under these conditions we again found no significant difference in the magnitude of the responses. In addition, the cytotoxic cells generated in response to uv induced tumors appeared to be highly cross reactive with respect to their killing potential. Cross reactive killing was observed between all uv induced tumors tested as well as with a syngeneic benz[a]pyrene (BP) induced fibrosarcoma. No cytotoxicity was observed against normal syngeneic PEC's even through these cells were shown to be susceptible to lysis by anti-H-2^k effector cells. It was concluded that: (a) A significant number of host-derived macrophages are present in uv tumor tissue. (b) These macrophages are important for the in vitro generation of tumor specific cytotoxicity. (c) Spleen cells from uv treated mice are capable of recognizing and responding against uv tumor associated antigens in vitro. Cytotoxic effector cells generated in response to uv induced tumors appear to have specificity for tumor associated antigens (TAA) present on all uv tumors tested as well as a syngeneic BP induced tumor. The relationship between in vivo and in vitro reactivity against uv tumors is discussed.     

Properties of ultraviolet light-induced suppressor lymphocytes within a syngeneic tumor system

It has been reported that ultraviolet light (uv) irradiation of normal C3Hf mice generates theta-positive suppressor lymphocytes which, as assayed by adoptive transfer studies, mediates susceptibility to transplanted syngeneic uv-induced tumors. We now report that the cells with suppressor activity can be recovered and enriched for using nylon wool columns. These suppressor cells are found in the nylon nonadherent fraction. Further, while uv irradiated mice are susceptible to supporting the growth of transplanted syngeneic uv tumors long after termination of the uv exposures, adoptively transferred suppression wanes and normal mice exhibit time-dependent recovery.     

Immunologic effects of whole-body ultraviolet (uv) irradiation. III Defective splenic adherent cell function in alloantigen-stimulated T-cell proliferation

The daily exposure of a mouse to ultraviolet (uv) radiation causes a selective depletion of Ia-bearing adherent cells in that animal's spleen. This depletion manifests itself in functional deficiencies in the presentation of protein antigens and haptens to T cells. The present studies demonstrate a defect in splenic adherent cells (SAC) from uv-irradiated mice resulting in defective alloantigen presentation. We show that unfractionated splenocytes and SAC from uv-irradiated mice show decreased stimulatory activity in allogeneic MLR. We then utilize this phenomenon induced by uv radiation to characterize the stimulator cell in the M locus (Mls) determinant-driven MLR. We show that the stimulator cell in Mls determinant-driven MLR is an adherent cell and demonstrate that this stimulator cell bears Ia determinants by showing that whole spleen cells and SAC from mice treated with uv radiation are inefficient stimulators of the Mls determinant-driven MLR. The importance of the Ia determinant on the stimulator cells in Mls determinant-driven MLR is corroborated by the demonstration that a monoclonal antibody directed at this determinant fully blocks the Mls determinant-driven MLR. The significance of these studies to the problem of alloreactions in vivo is discussed.     

Evidence for the generation of suppressor cells by ultraviolet radiation

The majority of murine skin tumor induced with ultraviolet (uv) light are unique in that they are of sufficient antigenicity to be consistently rejected when transplanted into normal syngeneic animals. However, the exposure of normal syngeneic mice to subcarcinogenic levels of uv prior to tumor transfer results in the progressive growth of transplanted uv-tumors. We report that normal mice can also be rendered tumor susceptible by the adoptive transfer of lymphoid cells from either tumor bearing or short term uv exposed donors. Further, the adoptive transfer of tumor susceptibility can be abolished by the pretreatment of cell suspensions from uv exposed donors with anti-theta and complement. These results suggest that uv irradiation may generate the development of T lymphocytes with suppressor activity.     

Cell-mediated immune response to syngeneic ultraviolet-induced tumors. II. The properties and antigenic specificities of cytotoxic T lymphocytes generated in vitro following removal from syngeneic tumor-immunized mice.

The immune responses of C3Hf mice to syngeneic fibrosarcomas induced with either ultraviolet light or methychlolanthrene (MCA) were measured in vitro by the ability of cytotoxic lymphocytes (CTL) from immunized animals to kill ⁵¹Cr-labeled tumor targets in a 6-hr assay. The CTL were generated by the in vitro culturing of draining popliteal lymph node (DLN) cells derived from animals that were footpad immunized 8 days previously. It was determined that CTL activity could be generated using DLN from both normal (uv tumorresistant) and uv-exposed (uv tumor-susceptible) C3H mice. The kinetics of CTL generation between these two groups, however, was different in that the lymphocytes from normal animals appeared to differentiate into CTL faster than the lymphocytes from the uv-irradiated mice. The in vitro generation of CTL activity was found to be extremely radiosensitive and was also inhibited by the presence of viable tumor cells within the cell culture. Once generated, it was observed that the CTL were extremely insensitive to the effects of gamma irradiation. It was also established that the CTL is a T lymphocyte that appears to be Ia^?. The CTL derived from mice immunized to syngeneic uv- or MCA-induced tumors were capable of expressing cross-reactive non-MHC-restricted killing of multiple tumor targets. Cold cell inhibition experiments confirmed the presence of cross-reactive determinants on various tumors and also established the presence within a single CTL preparation of effector cells with specificity for both the unique tumor specific transplantation antigens as well as the common (cross-reactive) tumor-associated antigens.     

Different types of thermoconditional clear plaque-mutants and prophage induction by heat or cold of Serratia phage kappa.

Phage kappa of Seratia marcescens was treated with different mutagens to induce thermoconditional clear plaque-mutants. 176 mutants obtained were analysed by crosses and found to be located in clear plaque-region III. Two types resembling the mutants t2 and t1 of phage lambda were identified. Lysogens for the mutant 126 can be induced by heat and even by cold whereas they are scarcely inducible by uv. Nevertheless, a 126 prophage like a uv inducible ct 163 prophage can be sensitized to thermoinduction by short preirradiation if the cells are incubated for 30 to 45 min between uv exposure and heating. With ct 163 this time corresponds to the minimum extension of latent period after uv induction compared with infection at low moi. A mutant of clear plaque-region II, c154, shows an inverted thermoconditional behaviour forming clear plaques at 30 degrees C and turbid plaques due to lysogenization at 37 degrees C.     

Axis determination in eggs of Xenopus laevis: A critical period before first cleavage,identified by the common effects of cold,pressure and ultraviolet irradiation

Exposure of eggs of Xenopus laevis to a temperature of 1.0°C for 4 min or a pressure of 8000 psi for 5 min in a critical period before first cleavage results in embryos exhibiting a reduction and loss of structures of the body axis. The deficiencies occur in a craniocaudal progression which is dose dependent. In the extreme, totally axis-deficient embryos with radial symmetry are formed. Maximum sensitivity to cold and pressure occurs at 0.6 of the time from fertilization to first cleavage and extends from approximately 0.4 to 0.8, the period between pronuclear contact and mitosis, and the approximate period of gray crescent formation. The effects of cold and pressure resemble those previously reported for uv irradiation in that (1) the types of axis-deficient embryos produced are morphologically indistinguishable; (2) sensitivity in all cases ends before 0.8; (3) cold and uv effects, although not those of pressure, can be prevented by cotreatment with D₂O; and (4) impaired eggs can be rescued by oblique orientation. We interpret these results as follows: during the 0.4–0.8 period the egg reorganizes its contents in a manner critical for subsequent development of the embryonic body axis. The reorganization process involves cytoskeletal elements, some of which are sensitive to cold, pressure, and uv, and protected by D₂O. Rescue by oblique orientation can be understood as the result of a gravity-driven reorganization of the egg's contents, supplanting the normal mechanochemical process impaired in treated eggs.     

Embryonic lethality and tumorigenesis caused by segmental aneuploidy on mouse chromosome 11.

Chromosome engineering in mice enables the construction of models of human chromosomal diseases and provides key reagents for genetic studies. To begin to define functional information for a small portion of chromosome 11, deficiencies, duplications, and inversions were constructed in embryonic stem cells with sizes ranging from 1 Mb to 22 cM. Two deficiencies and three duplications were established in the mouse germline. Mice with a 1-Mb duplication developed corneal hyperplasia and thymic tumors, while two different 3- to 4-cM deficiencies were embryonically lethal in heterozygous mice. A duplication corresponding to one of these two deficiencies was able to rescue its haplolethality.     

SYNTHESIS OF ETHANOLAMINE PHOSPHOGLYCERIDES BY MICROSOMES FROM THE BRAINS OF JIMPY AND QUAKING MICE

Abstract— —Brains of jimpy and quaking mice are known to be deficient in myelin and alkenylacyl-glycero-phosphorylethanolamines (alkenylacyl-GPE, ethanolamine plasmalogens). Ethanolamine plasmalogen synthetic activity appeared to be normal and ethanolamine phosphotransferase (EC 2.7.8.1) activities are higher in the brain microsomes from jimpy and quaking mice than in their littermate controls when the activities are assayed with alkylacylglycerols and CDP[¹⁴C]ethanolamine. When endogenous diradylglycerols were the substrate, the rate of synthesis of diacyl-GPE was normal but the rate of synthesis of the ether lipids, alkenylacyl-GPE and alkylacyl-GPE, was 33% and 8% below control levels for jimpy brain microsomes and quaking brain microsomes respectively. This difference is probably due to a normal content of diacylglycerols and a deficient content of alkylacylglycerols in the mutant brain microsomes. The apparent alkylacylglycerol deficiencies in the microsomes correspond with the ethanolamine plasmalogen deficiencies in the brains of these mutant mice.     

Immunological reconstitution of T-cell-deprived mice. I. Inability of thymosin to restore spleen cell mitogen and tumor allograft response.

Ultraviolet light (uv)-induced tumors appear to possess unique and common determinants, both of which can function as rejection antigens in appropriate in vivo tumor transplantation assays. We postulate that the common tumor antigens can play an important role in anti-tumor immunity. We have previously reported on the phenomenon of ultraviolet light generated suppressor lymphocytes which mediate susceptibility to transplanted uv-induced murine skin tumors. We now report that these uv generated suppressor cells are capable of specifically inhibiting effector responses directed against the common tumor antigens. A hypothesis is presented to explain how the presence of suppressor cells might account for the observed differences between in vivo and in vitro assays of tumor immunity.     

Tumor formation by SV40-transformed human cells in nude mice: the role of SV40 T antigens

Human cells transformed in vitro by SV40 rarely form tumors in nude mice. We examined whether these cells as a group are inherently nontumorigenic or whether they are potentially tumorigenic but rejected by the athymic host, possibly by nonspecific immune mechanisms. SV80 and NG8 are SV40-transformed human cell lines that express all of the transformed properties, including anchorage-independent growth, but do not form tumors in adult nude mice after injection of as many as 10(8) cells. Both the SV80 and NG8 cell lines have SV40-specific transplantation antigens which crossreact with those present on SV40-transformed (but tumorigenic) rodent cells. We found that SV80 cells, though not NG8 cells, induced progressively growing lethal tumors if the cells are injected repeatedly into neonatal nude mice. Somatic cell hybrids between SV80 or NG8 cells and a highly tumorigenic cell line derived from a human tumor continue to express the virus-induced antigens and fail to form tumors in adult nude mice. These results strongly suggest that at least for some SV40-transformed human cells, the failure to form tumors in nude mice may be due to their expression of virus-induced transplantation antigens rather than the absence of tumorigenic potential.     

Nucleotide excision repair- and p53-deficient mouse models in cancer research

Cancer is caused by the loss of controlled cell growth due to mutational (in)activation of critical genes known to be involved in cell cycle regulation. Three main mechanisms are known to be involved in the prevention of cells from becoming cancerous; DNA repair and cell cycle control, important to remove DNA damage before it will be fixed into mutations and apoptosis, resulting in the elimination of cells containing severe DNA damage. Several human syndromes are known to have (partially) deficiencies in these pathways, and are therefore highly cancer prone. Examples are xeroderma pigmentosum (XP) caused by an inborn defect in the nucleotide excision repair (NER) pathway and the Li-Fraumeni syndrome, which is the result of a germ line mutation in the p53 gene. XP patients develop skin cancer on sun exposed areas at a relatively early age, whereas Li-Fraumeni patients spontaneously develop a wide variety of early onset tumors, including sarcomas, leukemia's and mammary gland carcinomas. Several mouse models have been generated to mimic these human syndromes, providing us information about the role of these particular gene defects in the tumorigenesis process. In this review, spontaneous phenotypes of mice deficient for nucleotide excision repair and/or the p53 gene will be described, together with their responses upon exposure to either chemical carcinogens or radiation. Furthermore, possible applications of these and newly generated mouse models for cancer will be given.     

Ultraviolet Rays Induced Expression of Lectins on the Surface of a Squamous Carcinoma Keratinocyte Cell Line

Human keratinocytic cells from squamous carcinoma (SCL-1) present, under resting conditions, relatively low amounts of endogenous lectins (sugar-binding proteins). Upon uv irradiation, they express on their cell surface large amounts of endogenous lectin molecules able to bind neoglycoproteins bearing either α-l-rhamnosyl or α-d-glucosyl residues. A similar binding specificity was found with normal human keratinocytes under the same culture conditions. At sun-like doses, uv.A (365 nm) was more efficient than uv.B (312 nm) in the expression of such receptors on the surface of SCL-1 cells. The increased presentation of lectins by SCL-1 cells was transient and reached a maximum 4 h after irradiation. Such a specific modulation of receptor expression upon uv irradiation might be biologically significant, considering the numerous intercellular recognition phenomena in skin biology. α-l-Rhamnose-specific receptor on SCL-1 could not be distinguished from α-d-glucose-specific receptor on the basis of neoglycoproteins binding, uptake, and related inhibitions. Lectin expression was mainly detected on the cell surface, and its overexpression due to uv rays required ade novoprotein synthesis process.     

Wide spectrum of tumors in knock-in mice carrying a Cdk4 protein insensitive to INK4 inhibitors.

We have introduced a point mutation in the first coding exon of the locus encoding the cyclin-dependent kinase 4 (Cdk4) by homologous recombination in embryonic stem cells. This mutation (replacement of Arg24 by Cys) was first found in patients with hereditary melanoma and renders Cdk4 insensitive to INK4 inhibitors. Here, we report that primary embryonic fibroblasts expressing the mutant Cdk4R24C kinase are immortal and susceptible to transformation by Ras oncogenes. Moreover, homozygous Cdk4(R24C/R24C) mutant mice develop multiple tumors with almost complete penetrance. The most common neoplasia (endocrine tumors and hemangiosarcomas) are similar to those found in pRb(+/-) and p53(-/-) mice. This Cdk4 mutation cooperates with p53 and p27(Kip1) deficiencies in decreasing tumor latency and favoring development of specific tumor types. These results provide experimental evidence for a central role of Cdk4 regulation in cancer and provide a valuable model for testing the potential anti-tumor effect of Cdk4 inhibitors in vivo.     

Inhibition by ultraviolet light of pole cell formation in Smittia sp (Chironomidae,Diptera): Action spectrum and photoreversibility

The formation of pole cells (primordial germ cells) in Smittia sp can be inhibited by ultraviolet (uv) irradiation without causing significant mortality. Until 70 min after egg deposition, pole cells are suppressed by low uv doses applied to the posterior pole region. Microbeam irradiation of a target area including the oosome inhibits pole cell formation; this is not observed after irradiation of other target areas. The action spectrum for uv inhibition of pole cells shows a distinct peak at 260 nm; its shape suggests that a nucleic acid or nucleic acid-protein complex acts as an effective target. Independent evidence for the involvement of a nucleic acid moiety is derived from the fact that uv inhibition of pole cell formation is photoreversible. The results are discussed in the context of pole cell determination by localized cytoplasmic components.     

Alterations des fonctions de reproduction chez les souris males deficientes en recepteur aux estrogenes

In male mice, ER disruption causes infertility due to deficiencies in both gametogenesis and sexual behavior. An age dependent development of testicular dysmorphogenesis results in a progressive loss of germ cells. Circulating gonadotropins and testosterone are not appreciably higher in ERKO males, indicating that testicular androgens can effectively exert feedback on gonadotropins in the absence of aromatization and activation of ER. Infertility in male mice ERKOs does not appear to be a result of compromised androgen hormone action since the morphology of male reproductive tract organs is largely unaffected. Mounting behavior of ERKO males is normal whereas the ability to intromit and ejaculate is severely compromised.     

Visualization under ultraviolet light enhances 100-fold the sensitivity of peroxidase-stained blots

As described in this article, visualization and/or photography under uv light of 4-chloro-1-naphthol-developed, peroxidase-marked immunoblots allows an increase in sensitivity of more than 100 times over the apparent staining results observable under normal visible white light. This increase in sensitivity can be obtained with the minimal additional requirement of an uv lamp, with the actual chloronaphthol staining procedure remaining unaltered and thereby allowing the monitoring of specific reactions with much smaller quantities of antigen or antibodies. Substantial shortening of the procedure is another advantage, making it possible to complete in 20 min or even less a procedure usually requiring 3 to 6 h. The phenomenon depends on the uv absorption and the fluorescence quenching properties of the products of the peroxidase reaction. The absorption spectra of the membranes with or without peroxidase products indicate that an intermediate in the peroxidase reaction is responsible for the absorption under uv light. This intermediate accumulates under conditions where the final product absorbing in the visible light has not begun to be produced, thus explaining the large increase in sensitivity. The behaviors of three types of membranes, nitrocellulose, nylon, and Immobilon (PVDF), are compared. Due to its lower uv absorption, PVDF gives by far the best results, followed by nitrocellulose.     

Immunoprecipitation of pyrimidine(6-4)pyrimidone photoproducts and cyclobutane pyrimidine dimers in uv-irradiated DNA

Biological studies suggest that a significant proportion of the cytotoxicity observed in mammalian cells after uv irradiation may be due to damage other than cyclobutane dimers in DNA. Although pyrimidine-pyrimidone (6-4) photoproducts have been implicated as major contributors to cell lethality, their induction has been measured at considerably less than cyclobutane pyrimidine dimers when measured by chromatographic techniques. Because the yield of (6-4) photoproducts may be reduced by their lability to extreme heat and pH, we have advised an alternative, immunological quantification which does not require DNA hydrolysis. Affinity-purified rabbit antisera were used to precipitate low molecular weight 32P-labeled PM2 DNA irradiated with increasing fluences of uv light. DNA of known molecular weight was used to determine rates of induction for antibody-binding sites associated with (6-4) photoproducts and cyclobutane dimers. These rates were calculated to be 0.6 (6-4) photoproducts and 1.2 cyclobutane dimers/10(8) Da/J/m2. At low uv fluences (6-4) photoproducts were induced at one-half the rate of cyclobutane dimers, whereas at higher fluences (6-4) photoproducts predominated.     

Induction of protective immunity against Schistosoma mansoni by a nonliving vaccine. II. Response of mouse strains with selective immune defects

The efficacy of a new vaccination procedure against Schistosoma mansoni, involving intradermal injection of nonliving antigen combined with the bacterial adjuvant Mycobacterium bovis strain bacillus Calmette Guérin, was tested in several strains of mice. Development of protection against subsequent infection was compared with in vivo skin test reactivity and in vitro humoral reactivity to soluble and surface-associated schistosome antigens. Significant levels of resistance and immune response were displayed by many inbred mouse strains, including C57BL/6J, C3H/HeN, and CBA/J, as well as outbred Swiss-Webster mice. However, no definite correlation was observed between the level of any particular immune response and the level of resistance to challenge S. mansoni infection. Development of protective immunity was also examined in mice with various immune defects, to determine whether these responses are relevant to resistance in this model. Animals with defective specific immediate hypersensitivity response due to deficiencies in IgE (SJL/J) or mast cell (W/Wv) production displayed strong resistance as a result of immunization. Likewise, mice bearing the lpsd (C3H/HeJ) or xid (CBA/N) mutations, affecting cellular or humoral response to certain thymus-independent antigens, developed significant levels of resistance after immunization. A/J mice, with defects in cellular recognition of bacterial endotoxin as well as deficiencies in natural killer cell activity and complement function, also showed significant protective immunity. Thus, these reactivities do not appear to be essential to the resistance against S. mansoni induced by the nonliving vaccine. Two nonresponder strains were identified, P and BALB/c. P mice were defective in specific delayed hypersensitivity response as well as resistance to infection. However, BALB/c mice showed no obvious immune deficiencies at the time of challenge. These results agreed with previous findings in mice immunized by exposure to radiation-attenuated cercariae with one exception; BALB/c mice were protected by vaccination with irradiated cercariae but not by the nonliving vaccine. Thus, further examination of immune response in mice identified in this study as high and low responder strains should allow characterization of critical immune resistance mechanisms induced by the nonliving vaccine, as well as immune mechanisms operating in common between these two models of resistance to S. mansoni.     

Targeted expression of MYCN causes neuroblastoma in transgenic mice.

The proto-oncogene MYCN is often amplified in human neuroblastomas. The assumption that the amplification contributes to tumorigenesis has never been tested directly. We have created transgenic mice that overexpress MYCN in neuroectodermal cells and develop neuroblastoma. Analysis of tumors by comparative genomic hybridization revealed gains and losses of at least seven chromosomal regions, all of which are syntenic with comparable abnormalities detected in human neuroblastomas. In addition, we have shown that increases in MYCN dosage or deficiencies in either of the tumor suppressor genes NF1 or RB1 can augment tumorigenesis by the transgene. Our results provide direct evidence that MYCN can contribute to the genesis of neuroblastoma, suggest that the genetic events involved in the genesis of neuroblastoma can be tumorigenic in more than one chronological sequence, and offer a model for further study of the pathogenesis and therapy of neuroblastoma.