The response of L5178Y lymphoma sublines to oxidative stress: antioxidant defence, iron content and nuclear translocation of the p65 subunit of NF-kappaB

We examined the response to hydrogen peroxide of two L5178Y (LY) sublines which are inversely cross-sensitive to hydrogen peroxide and X-rays: LY-R cells are radio-resistant and hydrogen peroxide-sensitive, whereas LY-S cells are radiosensitive and hydrogen peroxide-resistant. Higher initial DNA breaks and higher iron content (potentially active in the Fenton reaction) were found in the hydrogen peroxide sensitive LY-R cells than in the hydrogen peroxide resistant LY-S cells, whereas the antioxidant defence of LY-R cells was weaker. In particular, catalase activity is twofold higher in LY-S than in LY-R cells. The content of monobromobimane-reactive thiols is 54% higher in LY-S than in LY-R cells. In contrast, the activity of glutathione peroxidase (GPx) is about two times higher in LY-R than in LY-S cells; however, upon induction with selenium the activity increases 15.6-fold in LY-R cells and 50.3-fold in LY-S cells. Altogether, the sensitivity difference is related to the iron content, the amount of the initial DNA damage, as well as to the efficiency of the antioxidant defence system. Differential nuclear translocation of p65-NF-kappaB in LY sublines is due to the more efficient antioxidant defence in LY-S than in LY-R cells.     

Differential induction of apoptosis in x-irradiated L5178Y sublines bearing p53 mutation

We examined apoptosis and expression of p53, E2F-1, bax, bclx_L and bcl2 proteins in two L5178Y (LY) murine lymphoma sublines, LY-R and LY-S, which differ in radiosensitivity and double-strand break (DSB) repair. Both sublines are heterozygous for a p53 mutation in codon 170 that precludes the transactivation function. Accordingly, there is no G1/S arrest after irradiation.We found that there is no change in expression of E2F-1, bax, bclx_L or bcl2 proteins in both LY sublines after x-irradiation. LY-R cells do not constitutively express bcl2, whereas both sublines show high bax content. Radiation induces delayed apoptosis to a greater extent in LY-S than in LY-R cells. The apoptosis can be seen 24 h after irradiation (2 Gy) of LY-S cells, with a maximum at 48 h. LY-R cells need 5 Gy and 72 h post-irradiation incubation to show marked apoptosis (identified by the TUNEL method). The reported observations support the assumption that differential radiosensitivity of LY sublines is associated with the induction of apoptosis that is not related to transactivation by p53 and is primarily related to differential DNA repair ability. Received: 19 August 1999 / Accepted in revised form: 30 November 1999     

Promotion of photodynamic therapy-induced apoptosis by stress kinases.

Photodynamic therapy (PDT), a cancer treatment that employs a photosensitizer and visible light, induces apoptosis in murine LY-R leukemic lymphoblasts and in CHO cells, but the rate and extent of apoptosis are much greater in LY-R cells. Three MAPK family members, ERK1/ERK2, SAPK/JNK, and p38/HOG, are important intermediates in signal transduction pathways. To ascertain whether activation of one or more MAPKs could mediate PDT-induced apoptosis, Western blot analysis has been performed on the proteins of LY-R and CHO cells at various times following lethal (90 - 99% cell kill) doses of PDT photosensitized by the phthalocyanine Pc 4. The blots were probed with antibodies to each of the proteins as well as antibodies specific for the activated (phosphorylated) forms of each kinase. Of the three MAPK types, only the p46 and p54 SAPK/JNKs were found to be activated by PDT in LY-R cells, with a maximum approximately threefold increase in the content of the phosphorylated forms reached in 30 - 60 min. An even larger relative activation was observed in CHO cells. PDT did not affect ERK and p38/HOG activation in LY-R cells. In the case of CHO cells, however, ERK2 was slightly activated at 5 min post-PDT, then declined, and p38/HOG was strongly activated from 5 to 60 min post-PDT. A specific inhibitor (PD98059) of MEK1, the kinase that activates ERK, had little or no effect on PDT-induced apoptosis in either LY-R or CHO cells. In contrast, a specific inhibitor of p38/HOG (SB202190) blocked PDT-induced apoptosis in LY-R cells with a lesser effect in CHO cells. The results suggest that both the SAPK and p38/HOG cascades can be stimulated by PDT and that the latter participates in both rapid and slow PDT-induced apoptosis. Furthermore, the high level of constitutively active p38/HOG in LY-R cells may poise those cells for rapid activation of apoptosis following PDT.     

干旱胁迫下小麦幼苗根、叶多胺含量和多胺氧化酶活性的变化

 以抗旱性不同的两个小麦品种（‘晋麦33’和‘温麦8’）（Triticum aestivum cv. Jinmai 33 and Wenmai 8）为材料,研究了干旱胁迫下多胺含量和多胺氧化酶活性的变化。结果表明：旱过程中,幼苗根、叶中腐胺（Put）、亚精胺（Spd）、精胺（Spm）3种多胺含量和多胺氧化酶（PAO）活性先迅速升高,而后下降。与抗旱性弱的‘晋麦33’相比,抗旱性强的品种‘温麦8’幼苗根、叶中Spd、Spm 含量初期升高幅度大,之后下降速率减慢;PAO活性的变化与之相反,‘晋麦33’的PAO活性提高的幅度大于‘温麦8号’。多胺含量和PAO活性在小麦幼苗的根与叶之间呈极显著正相关。干旱初期,小麦根、叶中多胺迅速积累可能是干旱胁迫反应的一个信号,随后较高的Spd、Spm 水平有利于增强小麦幼苗的抗旱性。     

Does ionizing radiation induce an adaptive response in mouse L5178Y lymphoma cells?]

Growth kinetics of LY-S and LY-R cells (radiosensitive and radioresistant sublines of murine lymphoma L5178Y) has been investigated after beta-irradiation at cumulative doses of 1.5 to 20 cGy and dose rates of 0.8-10 mGy/h. It has been found that after 48 h culture in a complete medium the number of cells differed 5 times, whereas after X- and gamma-irradiation, Do values differed 1.62 times. Using the growth rate as the end point in evaluating the combined effect of beta-irradiation (10 cGy) and subsequent X-irradiation with lethal doses, we observed an increased relative cell number, in comparison to that after X-irradiation alone (an "adaptive response", using this criterion), in LY-S cells irradiated with a dose of 2 Gy. In contrast, when reproductive death of LY-S and LY-R cells the end-point analyzed, the lethal effect of consecutive beta- and X-irradiation in LY-S cells was higher than that expected for X-radiation alone (the synergistic effect).     

Labile iron pool correlates with iron content in the nucleus and the formation of oxidative DNA damage in mouse lymphoma L5178Y cell lines

Labile iron pool (LIP) constitutes a crossroad of metabolic pathways of iron-containing compounds and is midway between the cellular need for iron, its uptake and storage. In this study we investigated oxidative DNA damage in relation to the labile iron pool in a pair of mouse lymphoma L5178Y (LY) sublines (LY-R and LY-S) differing in sensitivity to hydrogen peroxide. The LY-R cells, which are hydrogen peroxide-sensitive, contain 3 times more labile iron than the hydrogen peroxide-resistant LY-S cells. Using the comet assay, we compared total DNA breakage in the studied cell lines treated with hydrogen peroxide (25 microM for 30 min at 4 degrees C). More DNA damage was found in LY-R cells than in LY-S cells. We also compared the levels of DNA lesions sensitive to specific DNA repair enzymes in both cell lines treated with H(2)O(2). The levels of endonuclease III-sensitive sites and Fapy-DNA glycosylase-sensitive sites were found to be higher in LY-R cells than in LY-S cells. Our data suggest that the sensitivity of LY-R cells to H(2)O(2) is partially caused by the higher yield of oxidative DNA damage, as compared to that in LY-S cells. The critical factor appears to be the availability of transition metal ions that take part in the OH radical-generating Fenton reaction (very likely in the form of LIP).     

Lethal and mutagenic effects of radiation and alkylating agents on two strains of mouse L5178Y cells

In previous studies, the two closely related strains of L5178Y (LY) mouse lymphoma cells, LY-R and LY-S, have been shown to differ in their sensitivity to UV and ionizing radiation. Thus, in comparison to strain LY-R, strain LY-S has been found to be more sensitive to the lethal effects of ionizing radiation, more resistant to the lethal effects of UV radiation, but less mutable at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus by both UV and X-radiation. In the present work, the lethal and mutagenic effects of ethyl methanesulfonate (EMS), methyl nitrosourea (MNU) and UV radiation (254 nm) were compared in the two strains. Mutability at the Na+/K+-ATPase locus as well as the HGPRT locus was determined. As previously reported, we found strain LY-S to be more resistant than strain LY-R to the lethal effects of UV radiation. In contrast, strain LY-S was more sensitive to the cytotoxic effects of the two alkylating agents. In spite of these differences in sensitivity, we found strain LY-S to be less mutable than strain LY-R by all 3 agents at the HGPRT locus. At the Na+/K+-ATPase locus, strain LY-S was also less mutable than strain LY-R by equal concentrations of EMS and UV radiation and by equitoxic concentrations of MNU. However, the difference between the strains was much more pronounced at the HGPRT locus than at the Na+/K+-ATPase locus. We have suggested that the interaction of unrepaired lesions in strain LY-S tends to cause an excess of deletions and multilocus effects, which in turn result in a locus-dependent decrease in the recovery of viable LY-S mutant cells.     

PKA controls a level of topoisomerase I mRNA in mouse L5178Y lymphoma cells treated with db-cAMP

The level of topoisomerase I mRNA was measured in cells of two mouse lymphoma (LY) sublines treated with db-cAMP. A transient increase of the level was observed to be of about 60% of the basic level and to have maximum after the 3 h treatment of LY-S cells. The increase in LY-R subline was two-fold lower. The activity of PKA in a cytosol fraction of LY-S cells was 1.75 times higher than that in LY-R cells. The activity of PKA in membranes and nuclear fraction did not differ significantly in both cell types. When the activity of PKA in LY-S cells was inhibited with H8, no increase of the level of topoisomerase I mRNA was observed upon db-cAMP treatment of cells. We suggest that the activity of PKA in the cytosol controls the expression of topoisomerase I gene in LY cells at high concentration of cAMP.Abbreviations db-cAMP dibutyryl-cAMP - H8 N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide - LY mouse L5178Y lymphoma - PKA protein kinase A - topo I topoisomerase I     

Content of iron and copper in the nuclei and induction of pH 9-labile lesions in L5178Y sublines inversely cross-sensitive to H2O2 and x-rays

Cells from the L5178Y murine lymphoma subline LY-R are twice as resistent to killing by ionizing radiation than the subline LY-S. In contrast, LY-R cells are more sensitive to killing by H₂O₂, the effect being more pronounced at 37 °C than 0 °C. Initial DNA damage after H₂O₂ treatment (both temperatures, 5 min) has been estimated by the comet assay (single-cell gel electrophoresis) and fluorescent halo technique. According to both methods, the initial damage is significantly higher in LY-R cells, particularly that inflicted at O °C. Differences between DNA unwinding and rewinding abilities at pH 9 and 6.9 (estimated by the fluorescent halo technique) point to a considerable difference in pH-9-labile damage between the sublines, as observed previously for x-irradiated cells (Kapiszewska et al. 1992). In contrast to findings with x-irradiated cells, however, after H₂O₂ treatment this damage is more extensive in LY-R cells than in LY-S cells. Thus, the initial pH-9-labile damage corresponds to the pattern of sensitivity to H₂O₂ and x-rays. We suggest that this is caused by different proportions of cuprous and ferric ions found in the nuclei of LY sublines and by the different ability of these ions to react with H₂O₂ and water radiolysis products. The copper/iron ratio in the nucleus is 1.31 in LY-R cells and 4.84 in LY-S cells.     

Modulation of the effect of camptothecin in x-irradiated L5178Y-R and L5178Y-S cells by benzamide

The L5178Y (LY) murine lymphoma subline, LY-R, is more radioresistant and more sensitive to camptothecin (CPT, inhibitor of topisomerase I) than the second subline used in our investigation, LY-S. Post-irradiation treatment with 3 μM CPT enhanced the radiosensitivity of LY-S cells (D₀ decrease from 0.52 to 0.34 Gy), but did not change it in LY-R cells. Treatment with 2 mM benzamide [BZ, inhibitor of poly(ADP-ribosylation)] before x-rays and CPT increased the radiosensitivity of LY-R cells (D₀ decrease from 1.15 to 0.52) without further modification of radiosensitivity of LY-S cells. Activity of topoisomerase I was diminished 10 min after x-irradiation (5 Gy) in LY-S, but not in LY-R cells. The data on DNA damage (fluorescent halo or comet assays) showed that the ultimate fate of the cells did not depend on the DNA damage pattern estimated immediately after treatment (e. g. the damage was greater in x-rays plus CPT than in BZ plus x-rays plus CPT treated LY-R cells, although the radiosensitivity was less). Aphidicolin (inhibitor of DNA polymerases α and δ) applied concomitantly with CPT in cells not pre-treated with BZ prevented the increase in DNA damage in LY-R cells, but was without effect in LY-S cells. Taking into account the differential inhibition by x-rays of DNA synthesis in LY sublines and its reversion by BZ in LY-S but not in LY-R cells, we conclude that the pattern of DNA damage observed by the methods applied depended on the status of DNA replication. Received: 28 November 1995 / Accepted in revised form: 20 April 1996     

Chelating of iron and copper alters properties of DNA in L5178Y cells, as revealed by the comet assay.

We have previously found different proportions of iron and copper in nuclei of two sublines of murine lymphoma L5178Y (LY) and proposed a model of chromatin organization with these metal ions at the DNA attachment sites. We now examine the effect of chelators, desferal (DFO, iron-specific) and neocupreine (NEO, copper-specific) on DNA of LY-R and LY-S cells, using the comet and micronuclei frequency tests. There is less copper and more iron in LY-R nuclei than in LY-S nuclei. Accordingly, the effect of NEO is more marked in LY-R than in LY-S cells and in both sublines it is expressed as enhanced tail moment (measure of DNA damage in the comet assay) and increased micronuclei frequency. On the contrary, the effect of DFO on the tail moment is less pronounced in LY-R than in LY-S cells. With increasing DFO concentrations, there is a gradual decrease in the tail moment values below the control level in LY-S cells. In LY-R cells the tail moment values initially increase, then gradually decrease, eventually falling below the control level. This points to a dramatic conformational change that masks the effect of DNA discontinuities. The presence of the latter is indicated by the increase in micronuclei frequency. These results support the postulated differential role of iron and copper ions in maintaining the higher order DNA structure in LY sublines.     

Bridging the gap between plant and mammalian polyamine catabolism: a novel peroxisomal polyamine oxidase responsible for a full back-conversion pathway in Arabidopsis

In contrast to animals, where polyamine (PA) catabolism efficiently converts spermine (Spm) to putrescine (Put), plants have been considered to possess a PA catabolic pathway producing 1,3-diaminopropane, Delta(1)-pyrroline, the corresponding aldehyde, and hydrogen peroxide but unable to back-convert Spm to Put. Arabidopsis (Arabidopsis thaliana) genome contains at least five putative PA oxidase (PAO) members with yet-unknown localization and physiological role(s). AtPAO1 was recently identified as an enzyme similar to the mammalian Spm oxidase, which converts Spm to spermidine (Spd). In this work, we have performed in silico analysis of the five Arabidopsis genes and have identified PAO3 (AtPAO3) as a nontypical PAO, in terms of homology, compared to other known PAOs. We have expressed the gene AtPAO3 and have purified a protein corresponding to it using the inducible heterologous expression system of Escherichia coli. AtPAO3 catalyzed the sequential conversion/oxidation of Spm to Spd, and of Spd to Put, thus exhibiting functional homology to the mammalian PAOs. The best substrate for this pathway was Spd, whereas the N(1)-acetyl-derivatives of Spm and Spd were oxidized less efficiently. On the other hand, no activity was detected when diamines (agmatine, cadaverine, and Put) were used as substrates. Moreover, although AtPAO3 does not exhibit significant similarity to the other known PAOs, it is efficiently inhibited by guazatine, a potent PAO inhibitor. AtPAO3 contains a peroxisomal targeting motif at the C terminus, and it targets green fluorescence protein to peroxisomes when fused at the N terminus but not at the C terminus. These results reveal that AtPAO3 is a peroxisomal protein and that the C terminus of the protein contains the sorting information. The overall data reinforce the view that plants and mammals possess a similar PA oxidation system, concerning both the subcellular localization and the mode of its action.     

Biosynthesis profile and endogenous titers of polyamines differ in totipotent and recalcitrant plant protoplasts

The expression of totipotency in plant protoplasts is a complex developmental phenomenon and is affected by genetic and physiological factors. Polyamines (PAs) are known to be involved in a variety of growth and developmental processes in higher plants, as well as in adaptation to stresses. In this study, we present the homeostatic characteristics of the endogenous PA putrescine (Put), spermidine (Spd), and spermine (Spm) in totipotent (T) and non-totipotent (NT) tobacco protoplasts and in recalcitrant (R) grapevine protoplasts. T-tobacco protoplasts, with high division rates, have the highest level of endogenous PAs. In these protoplasts, the soluble-hydrolyzed fraction predominates and increases, and the insoluble-hydrolyzed fraction also increases, whereas soluble (S) PAs decrease rapidly during culture. The isolation process contributes to the increased Put levels, which are higher in freshly isolated NT-tobacco protoplasts than in T-protoplasts. During culture, total Put predominates over Spd and Spm, and the highest accumulation is found in T-protoplasts. Ornithine decarboxylase and arginase activities both increase in T-protoplasts, whereas arginine decarboxylase activity causes Put accumulation in NT-tobacco protoplasts. R-grapevine protoplasts show a different PA profile, mostly due to the lower PA content, the higher S-fraction, and the higher ratio of Spm to total PAs. The data suggest that the levels and metabolism of the intracellular PAs could be related to the expression of totipotency of plant protoplasts.     

An evaluation of polyamine immunocytochemistry using immunocytochemical model systems

Polyamine (PA) immunocytochemistry (ICC) was evaluated by a recently developed enzyme-linked immunosorbent assay (ELISA) binding test (ELISABT) using an anti-spermine (Spm) serum raised against Spm conjugated via the cross-linker N-(-maleimidobutyryloxy)succinimide (GMBS) with bovine serum albumin. In the test the antiserum showed strong immunoreactivity with N ¹-acetylspermine (Ac-Spm) and acetylspermidine (N ¹-Ac-Spd and N ⁸-Ac-Spd), and low immunoreactivity with Spm and Spd, which was, however, markedly enhanced after reaction with GMBS, acetic anhydride or glutaraldehyde. Complete agreement with results of immunoblot analysis was observed. PA-like immunoreactivity observed in the present PA ICC in cells in the foveolae and isthmus of rat gastric glands was completely abolished by absorption of the serum with N ¹,N ¹²-diacetyl-Spm, Ac-Spm or N ¹-Ac-Spd, but not by Spm or Spd. This absorption test was then simulated by an ELISA inhibition test (ELISAIT) with a solid phase conjugated with Ac-Spm or Spm, and by a PA ICC model system using Sepharose gel beads conjugated with each of several PAs. The results strongly suggest that the immunostaining in the gastric mucosa was mainly due to antibody species in the serum specific to acylated Spm and Spd, but not to Spm or Spd. Acetyl PAs exist at such low concentrations in animal tissues that they are virtually undetectable by current ICC methods. Therefore Spm and Spd are likely candidates for those detected, after having been converted by fixation into such PA derivatives as become reactive with the antiserum.     

Nuclear translocation of the p65 subunit of NF-κB in L5178Y sublines differing in antioxidant defense

We examined the induction of nuclear translocation of the p65 subunit of NF-κB in L5178Y (LY) cells. We used two LY sublines which are inversely cross-sensitive to hydrogen peroxide and x-rays: LY-R cells are radioresistant and oxidant-sensitive, whereas LY-S cells are radiosensitive and oxidant-resistant. Hydrogen peroxide, phorbol ester and x-rays caused a marked translocation of p65-NF-κB in LY-R cells and a weak translocation in LY-S cells. By manipulating the antioxidant defense status, we obtained an alteration in the p65-NF-κB translocation induction in LY-R cells. A similar effect was achieved with lovastatin pretreatment (25 μM, 24 h, 37°C). The response of LY-S cells under all these conditions was considerably weaker. We conclude that differential nuclear translocation of p65-NF-κB in LY sublines is not related to the lethal effect of the activating, damaging agent; rather it is due to the more efficient antioxidant defense in LY-S than in LY-R cells. Received: 10 November 1998 / Accepted in revised form: 2 March 1999     

Metabolic correlations of glucocorticoids and polyamines in inflammation and apoptosis

Glucocorticoid hormones (GC) are essential in all aspects of human health and disease. Their anti-inflammatory and immunosuppressive properties are reasons for therapeutic application in several diseases. GC suppress immune activation and uncontrolled overproduction and release of cytokines. GC inhibit the release of pro-inflammatory cytokines and stimulate the production of anti-inflammatory cytokines. Investigation of GC’s mechanism of action, suggested that polyamines (PA) may act as mediators or messengers of their effects. Beside glucocorticoids, spermine (Spm) is one of endogenous inhibitors of cytokine production. There are many similarities in the metabolic actions of GC and PA. The major mechanism of GC effects involves the regulation of gene expression. PA are essential for maintaining higher order organization of chromatin in vivo. Spermidine and Spm stabilize chromatin and nuclear enzymes, due to their ability to form complexes with negatively charged groups on DNA, RNA and proteins. Also, there is an increasing body of evidence that GC and PA change the chromatin structure especially through acetylation and deacetylation of histones. GC display potent immunomodulatory activities, including the ability to induce T and B lymphocyte apoptosis, mediated via production of reactive oxygen species (ROS) in the mitochondrial pathway. The by-products of PA catabolic pathways (hydrogen peroxide, amino aldehydes, acrolein) produce ROS, well-known cytotoxic agents involved in programmed cell death (PCD) or apoptosis. This review is an attempt in the better understanding of relation between GC and PA, naturally occurring compounds of all eukaryotic cells, anti-inflammatory and apoptotic agents in physiological and pathological conditions connected to oxidative stress or PCD.     

The repair of potentially lethal damage and sublethal damage in strains of mouse L5178Y lymphoma cells differing in radiation sensitivity

Mouse lymphoma strains L5178Y-R (LY-R) and L5178Y-S (LY-S), which are differentially sensitive to the cytotoxic effects of ionizing radiation, were found to differ in their abilities to repair potentially lethal damage (PLD) and sublethal damage (SLD). The results showed that strain LY-R was more proficient than strain LY-S in the repair of SLD. The split dose recovery observed in strain LY-S could be accounted for by its recovery during postirradiation incubation. In contrast, SLD repair occurred in the absence of PLD repair in strain LY-R. The possibility that the repair of PLD might be completed prior to the postirradiation incubation in strain LY-R was suggested by the decreased survival observed when the cells were irradiated in a hypotonic solution. The repair of PLD and SLD in strain LY-S was temperature sensitive, occurring during postirradiation incubations between 15 and 34 degrees C, but not at 37 or 40 degrees C. This temperature sensitivity is very similar to the temperature sensitivity of the repair of pH 9.6-labile lesions in DNA in strain LY-S, as reported previously. Thus postirradiation cellular recovery processes in strain LY-S may involve the repair of pH 9.6-labile lesions in DNA. Temperature-dependent changes in the postirradiation distribution of cells throughout the cell cycle were observed which could contribute to the temperature sensitivity of the postirradiation recovery of strain LY-S.     

Augmentation of the therapeutic efficacy of adoptive tumor immunotherapy by in vivo administration of slowly released recombinant interleukin 2

Summary Immunization of C57BL/6 mice with MMC-treated syngeneic lymphoma cells, MBL-2, caused the generation of antitumor effector cells in vivo and the immunized mice permanently rejected viable MBL-2 lymphoma cells. Both plastic nonadherent T cells and plastic adherent MØ obtained from MBL-2 immunized mouse peritoneal exudate cells revealed strong cytotoxic activity against MBL-2 lymphoma cells, whereas immune spleen cells were not highly active against MBL-2 lymphoma cells in vitro. However, systemic adoptive transfer of immune spleen cells into the MBL-2-bearing mice by i.v. infusion in conjunction with i.p. cyclophosphamide (100 mg/kg) treatment cured the mice of tumor. This therapeutic efficacy of immune spleen cells was reflected by the number of transferred effector cells and over 5×10⁷ immune spleen cells were required to cure the mice completely. The cells mediating in vivo rejection of MBL-2 lymphoma cells were Thy 1.2⁺ T cells. This ACIT was specific against MBL-2 lymphoma cells and had no effect on the growth of other syngeneic tumors, B16 melanoma or BMC6A fibrosarcoma. In vivo administration of recombinant interleukin 2 (r-IL 2) combined with ACIT greatly modulated the cure rate of tumor-bearing mice. In addition, we found that slowly released r-IL 2 administratered from an ALZET miniosmotic pump was more effective in augmenting the therapeutic efficacy of immune spleen cells in ACIT than a single injection of the same total dose of r-IL 2.     

Immunocytochemical localization of polyamines in the gastrointestinal tracts of rats and mice

An immunocytochemical method using a recently produced monoclonal antibody (ASPM-29) with an antibody specificity to spermine (Spm) and spermidine (Spd) fixed in situ, was used to demonstrate an immunocytochemical localization of polyamine (PA) pools in the gastrointestinal tracts of rats and mice. High PA immunoreactivity was always found in the cytoplasm of cells not only at the cell proliferative zone or the precursor cell zone but also at the neighboring non-proliferative premature cell zone of the epithelium, and a gradient of decreasing PA levels was noticed from these cells to the fully mature differentiated gastric surface mucous cells and absorptive cells of the small and large intestines. Also, strong staining for PAs was seen in the cytoplasm of fully differentiated gastric chief cells and neurons of both the myenteric and submucous plexuses, whereas the nuclei of the cells remained virtually unstained. These results may suggest that PAs are closely associated with the high biosynthetic activity in the cells of the gastrointestinal mucosa of normal rats and mice. This seems to be consistent with the PA imunocytochemical results previously obtained for neoplastic cells and active protein- or peptide-secreting cells, including exocrine or endocrine cell types.