S49 Mouse Lymphoma Cells Research Articles

CCMP and cUMP in Apoptosis: Concepts and Methods.

The cyclic nucleotides cAMP and cGMP are well-characterized second messenger molecules regulating many important intracellular processes, such as differentiation, proliferation, and apoptosis. The latter is a highly regulated process of programmed cell death wherein several regulatory proteins, like those belonging to the Bcl-2 family, are involved. The initiation of apoptosis is regulated by three different pathways: the intrinsic or mitochondrial, the extrinsic, and the ER stress pathway. Recently, it has been published that the pyrimidine cyclic nucleotides cCMP and cUMP also function as second messenger molecules, and additionally have an effect on apoptosis signaling pathways. cCMP induced PKA-independent apoptosis via the intrinsic and ER-stress pathway in S49 mouse lymphoma cells, and cCMP as well as cUMP induced apoptosis in human HEL cells via the intrinsic pathway. However, in human K-562 cells, which are known to be multidrug-resistant, cCMP and cUMP had no effect. Summarized in this chapter are the initiation of apoptosis by cCMP and cUMP regarding the various apoptotic pathways, the enzymes involved in apoptosis, as well as the most relevant methods for the detection and examination of apoptosis and the corresponding signaling pathways.

CCMP causes caspase-dependent apoptosis in mouse lymphoma cell lines

cCMP is a cyclic pyrimidine nucleotide which binds to and activates cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). In S49 lymphoma cells, cAMP induces apoptosis via PKA. In our present study, we examined the effect of cCMP on apoptosis in S49 mouse lymphoma cells and in PKA-deficient S49kin−cells. These two cell lines also lack PKG, hyperpolarization-activated cyclic nucleotide-gated channels 2 and 4 (HCN2 and HCN4) as assessed by real-time PCR. The cell-permeable analog cCMP–AM induced PKA- and PKG-independent apoptosis in S49 cells. In contrast, exchange protein activated by cAMP (Epac) activation did not induce apoptosis. cCMP induced caspase-dependent apoptosis via the intrinsic pathway, led to cytochrome c release from mitochondria and also activated the ER stress pathway. On the contrary, the extrinsic apoptotic pathway was not involved. Autophagy was not detectable after treatment with cCMP–AM in both cell lines. cAMP–AM, cGMP–AM, cUMP–AM as well as the cyclic nucleotides lacking the acetoxymethylester (AM)-group had no effect. cCMP–AM altered gene expression of the apoptotic-relevant gene Gadd45α and the immediate early response genes cFos and Nr4A1 in S49 wild-type (wt) cells. In conclusion, cCMP induces apoptosis of S49 lymphoma cells, independently of hitherto known cCMP target proteins.

Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A2 using cells deficient in the expression of scramblase

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A2 but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A2. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A2. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A2, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.

Investigation into the Role of Phosphatidylserine in Modifying the Susceptibility of Human Lymphocytes to Secretory Phospholipase A2 using Cells Deficient in the Expression of Scramblase.

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A2 but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt’s lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (87%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A2. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100 s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A2. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A2, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.

Phosphoinositide phosphatase SHIP-1 regulates apoptosis induced by edelfosine, Fas ligation and DNA damage in mouse lymphoma cells

S49 mouse lymphoma cells undergo apoptosis in response to the ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), FasL (Fas ligand) and DNA damage. S49 cells made resistant to ALP (S49(AR)) are defective in sphingomyelin synthesis and ALP uptake, and also have acquired resistance to FasL and DNA damage. However, these cells can be re-sensitized following prolonged culturing in the absence of ALP. The resistant cells show sustained ERK (extracellular-signal-regulated kinase)/Akt activity, consistent with enhanced survival signalling. In search of a common mediator of the observed cross-resistance, we found that S49(AR) cells lacked the PtdIns(3,4,5)P(3) phosphatase SHIP-1 [SH2 (Src homology 2)-domain-containing inositol phosphatase 1], a known regulator of the Akt survival pathway. Re-sensitization of the S49(AR) cells restored SHIP-1 expression as well as phosphoinositide and sphingomyelin levels. Knockdown of SHIP-1 mimicked the S49(AR) phenotype in terms of apoptosis cross-resistance, sphingomyelin deficiency and altered phosphoinositide levels. Collectively, the results of the present study suggest that SHIP-1 collaborates with sphingomyelin synthase to regulate lymphoma cell death irrespective of the nature of the apoptotic stimulus.

Fas/CD95 down-regulation in lymphoma cells through acquired alkyllysophospholipid resistance: partial role of associated sphingomyelin deficiency

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) induces apoptosis in S49 mouse lymphoma cells. A variant cell line, S49AR, made resistant to ALP, was found previously to be impaired in ALP uptake via lipid-raft-mediated endocytosis. In the present paper, we report that these cells display cross-resistance to Fas/CD95 ligation [FasL (Fas ligand)], and can be gradually resensitized by prolonged culturing in the absence of ALP. Fas and ALP activate distinct apoptotic pathways, since ALP-induced apoptosis was not abrogated by dominant-negative FADD (Fas-associated protein with death domain), cFLIP(L) [cellular FLICE (FADD-like interleukin 1beta-converting enzyme)-inhibitory protein long form] or the caspase 8 inhibitor Z-IETD-FMK (benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone). ALP-resistant cells showed decreased Fas expression, at both the mRNA and protein levels, in a proteasome-dependent fashion. The proteasome inhibitor MG132 partially restored Fas expression and resensitized the cells to FasL, but not to ALP. Resistant cells completely lacked SM (sphingomyelin) synthesis, which seems to be a unique feature of the S49 cell system, having very low SM levels in parental cells. Lack of SM synthesis did not affect cell growth in serum-containing medium, but retarded growth under serum-free (SM-free) conditions. SM deficiency determined in part the resistance to ALP and FasL. Exogenous short-chain (C12-) SM partially restored cell-surface expression of Fas in lipid rafts and FasL sensitivity, but did not affect Fas mRNA levels or ALP sensitivity. We conclude that the acquired resistance of S49 cells to ALP is associated with down-regulated SM synthesis and Fas gene transcription and that SM in lipid rafts stabilizes Fas expression at the cell surface.

Lipid rafts and metabolic energy differentially determine uptake of anti-cancer alkylphospholipids in lymphoma versus carcinoma cells

Perifosine is a member of the class of synthetic alkylphospholipids (APLs) and is being evaluated as anti-cancer agent in several clinical trials. These single-chain APLs accumulate in cellular membranes and disturb lipid-dependent signal transduction, ultimately causing apoptosis in a variety of tumor cells. The APL prototype edelfosine was previously found to be endocytosed by S49 mouse lymphoma cells via lipid rafts. An edelfosine-resistant cell variant, S49 AR, was found to be cross-resistant to other APLs, including perifosine. This resistance was due to defective synthesis of the raft constituent sphingomyelin, which abrogated APL cellular uptake. Sensitivity of S49 cells to edelfosine was higher than perifosine, which correlated with a relatively higher uptake. Human KB epidermal carcinoma cells were much more sensitive to APLs than S49 cells. Their much higher APL uptake was highly dependent on intracellular ATP and ambient temperature, and was blocked by chlorpromazine, independent of canonical endocytic pathways. We found no prominent role of lipid rafts for APL uptake in these KB cells; contrary to S49 AR cells, perifosine-resistant KBr cells display normal sphingomyelin synthesis, whereas APL uptake by the responsive KB cells was insensitive to treatment with methyl-β-cyclodextrin, a cholesterol-sequestrator and inhibitor of raft-mediated endocytosis. In conclusion, different mechanisms determine APL uptake and consequent apoptotic toxicity in lymphoma versus carcinoma cells. In the latter cells, APL uptake is mainly determined by a raft- and endocytosis-independent process, but metabolic energy-dependent process, possibly by a lipid transporter.

Both Sphingomyelin Synthases SMS1 and SMS2 Are Required for Sphingomyelin Homeostasis and Growth in Human HeLa Cells

Sphingomyelin (SM) is a vital component of cellular membranes in organisms ranging from mammals to protozoa. Its production involves the transfer of phosphocholine from phosphatidylcholine to ceramide, yielding diacylglycerol in the process. The mammalian genome encodes two known SM synthase (SMS) isoforms, SMS1 and SMS2. However, the relative contributions of these enzymes to SM production in mammalian cells remained to be established. Here we show that SMS1 and SMS2 are co-expressed in a variety of cell types and function as the key Golgi- and plasma membrane-associated SM synthases in human cervical carcinoma HeLa cells, respectively. RNA interference-mediated depletion of either SMS1 or SMS2 caused a substantial decrease in SM production levels, an accumulation of ceramides, and a block in cell growth. Although SMS-depleted cells displayed a reduced SM content, external addition of SM did not restore growth. These results indicate that the biological role of SM synthases goes beyond formation of SM.

Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin- and cholesterol-deficiency in lipid rafts

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway. The reason for this uptake failure is not understood. In the present study, we show that S49AR cells are unable to synthesize SM (sphingomyelin) due to down-regulated SMS1 (SM synthase 1) expression. In parental S49 cells, resistance to ALP could be mimicked by small interfering RNA-induced SMS1 suppression, resulting in SM deficiency and blockage of raft-dependent internalization of ALP and induction of apoptosis. Similar results were obtained by treatment of the cells with myriocin/ISP-1, an inhibitor of general sphingolipid synthesis, or with U18666A, a cholesterol homoeostasis perturbing agent. U18666A is known to inhibit Niemann-Pick C1 protein-dependent vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane. U18666A reduced cholesterol partitioning in detergent-resistant lipid rafts and inhibited SM synthesis in S49 cells, causing ALP resistance similar to that observed in S49AR cells. The results are explained by the strong physical interaction between (newly synthesized) SM and available cholesterol at the Golgi, where they facilitate lipid raft formation. We propose that ALP internalization by lipid-raft-dependent endocytosis represents the retrograde route of a constitutive SMS1- and lipid-raft-dependent membrane vesicular recycling process.

Anti-proliferative effects of 8-chloro-cAMP and other cAMP analogs are unrelated to their effects on protein kinase A regulatory subunit expression.

Conflicting reports have attributed 8-chloro-cAMP (Cl-cAMP)-mediated inhibition of tumor cell growth to either a toxic 8-chloro-adenosine (Cl-AdR) breakdown product or a Cl-cAMP-mediated decrease in ratio of Type I to Type II regulatory (R) subunits of protein kinase A (PKA). Using the MCF-7 human breast cancer and S49 mouse lymphoma cell lines as models, we show that the effects of Cl-cAMP and other cAMP analogs on growth and R subunit expression are unrelated. MCF-7 cell growth was insensitive to most analogs and inducers of cAMP, but was potently inhibited by Cl-cAMP acting through uptake and phosphorylation of its Cl-AdR breakdown product. Possible roles of adenosine receptors or P(2) purinoceptors in these Cl-cAMP-mediated growth effects were ruled out by studies with agonists and antagonists. Cholera toxin markedly decreased the ratio of Type I to Type II R subunits in MCF-7 cells without affecting growth, while growth inhibitory concentrations of Cl-cAMP or Cl-AdR had insignificant effects on this ratio. In S49 cells, where PKA activation is known to inhibit cell growth, PKA-deficient mutants retained sensitivity to both Cl-cAMP and the related 8-bromo-cAMP. Adenosine kinase (AK)-deficient S49 cells were inhibited only by higher concentrations of these 8-halogenated cAMP analogs. Of the commonly used cAMP analogs, only 8-(4-chlorophenylthio)-cAMP acted purely as a cyclic nucleotide-having no effect on PKA-deficient cells, but strongly inhibiting both wild-type and AK-deficient cells. Where growth inhibitory concentrations of most cAMP analogs reduced RI expression in the AK-deficient mutant, a functionally equivalent concentration of (N(6), O(2'))dibutyryl-cAMP maintained or increased this expression.

Differential Expression of Intracisternal A-Particle Transcripts in Immunogenic versus Tumorigenic S49 Murine Lymphoma Cells

Tumorigenic S49 mouse lymphoma cells (T-25) were compared to their nontumorigenic (immunogenic) substrate-adherent descendants (T-25-Adh), using the differential display technique. A 784-bp fragment with 92% sequence homology to the intracisternal A-particle (IAP) element family was isolated from the latter cells. IAP sequences are endogenous, noninfectious retroviral elements that can undergo transpositions and act as mutagens. Expression of IAP transcripts (as detected by the isolated fragment) was 5- to 10-fold higher in T-25-Adh cells than in T-25 cells. IAP RT-PCR cDNA clones derived from the immunogenic T-25-Adh cells, but not from T-25 cells, contain two distinctive motifs: (i) a motif characteristic of IAP elements expressed in lymphoid cells (lymphocyte specific, LS); (ii) a nonapeptide sequence known to stimulate cytotoxic T lymphocytes in a leukemia cell line expressing IAP sequences. In addition, expression of transcripts containing these motifs is enhanced in the immunogenic cells as opposed to the tumorigenic cells. Furthermore, one of the IAP elements (belonging to the LS1 subfamily) is specifically hypomethylated in the DNA of the immunogenic cells. The above-mentioned relationship was strengthened when tumorigenic revertants derived from T-25-Adh cells, as well as independently selected tumorigenic and immunogenic S49 sublines, were studied. In all cases, enhanced immunogenicity was linked to the up-regulation of specific IAP elements. No transpositions of LS1 elements were observed among the different sublines studied. These findings suggest that, in the S49 lymphoma, selectively expressed IAP retroviral elements may function in a tumor suppressive capacity by affecting the immunogenic potential of these cells.

On the spontaneous mutability of CpG sites in cultured S49 mouse lymphoma cells.

The frequent occurrence of human mutations at CpG dinucleotide sites has been attributed to cytosine methylation and hydrolytic deamination of the resulting 5-methylcytosine residue. Previously, we reported an unusually strong hotspot for spontaneous transitions at a CpG site in the gene for regulatory (R) subunit of protein kinase A in S49 mouse lymphoma cells. Now, using polymerase chain reaction-based methods to screen mutant populations for mutations at particular CpG sites, we show that two methylated CpG sites in the gene for hypoxanthine phosphoribosyltransferase are much less mutable than the R subunit hotspot site, suggesting that different methylated CpG sites are differentially susceptible to spontaneous mutation. We also present data on spontaneous R subunit mutations in cloned populations of 5-azacytidine-treated S49 cells that had been demethylated at the hotspot site in both R subunit alleles. Of 13 independent mutants isolated from populations grown from fully demethylated cells, seven had the hotspot mutation. We conclude that CG-->TA mutations at strong CpG hotspots do not require prior methylation of CpG sites.

Serum and Insulin Induce a Grb2-Dependent Shift in Agonist Affinity of β-Adrenergic Receptors

β-Adrenergic receptors transduce catecholamine binding to activation of adenylylcyclase, a response counter-regulated by insulin. Insulin stimulates tyrosine phosphorylation of Tyr 350/354, which abolishes the catecholamine response. Phosphorylation of Try 350/354 creates a Src homology 2 (SH2) domain on the β 2-adrenergic receptor and the binding of adaptor protein Grb2 to this SH2 domain of the β-adrenergic receptor takes place in an insulin-dependent manner. In membranes from serum-deprived S49 mouse lymphoma cells, GTPγS yields the well-known agonist-specific shift in agonist affinity for β 2-adrenergic receptors. The agonist-specific shift is observed in cell membranes either in the absence or in the presence of exogenously added purified Grb2. In membranes for serum-fed cells, in contrast, the addition of Grb2 induces an agonist-specific shift in receptor affinity that mimics addition of GTPγS to the membranes. The ability of the Grb2 to induce an agonist-specific shift in the membranes from serum-fed cells was abolished equally effectively either by competition with phosphopeptide harbouring the (p)YVNV motif or by disruption of the SH2 domain of added Grb2. Challenging Chinese hamster ovary cells with insulin (100 nM) for 30 min enabled Grb2 to induce an agonist-specific shift in agonist affinity for β 2-adrenergic receptors, suggestive of uncoupling of the receptors from G proteins. The insulin-dependent Grb2 effect on receptor–G-protein coupling was sensitive to competition by the pYVNY phosphopeptide and to disruption of the SH2 domain of Grb2. These data provide a biochemical link between the ability of insulin to counter-regulate catecholamine stimulation of cyclic AMP accumulation and the phosphorylation of the β-adrenergic receptor, consequent biding of the adaptor molecule Grb2 and disruption of receptor–G-protein coupling.

Analysis of the Gs/Mitogen-activated Protein Kinase Pathway in Mutant S49 Cells

Heterotrimeric G protein-coupled receptors can activate the mitogen-activated protein kinase (MAPK) cascade. Recent studies using pharmacological inhibitors or dominant-negative mutants of signaling molecules have advanced our understanding of the pathways from G protein-coupled receptors to MAPK. However, molecular genetic analysis of these pathways is inadequate in mammalian cells. Here, using the well characterized Gsalpha- and protein kinase A-deficient S49 mouse lymphoma cells, we provide the molecular genetic evidence that Gsalpha is responsible for transducing the beta-adrenergic receptor signal to MAPK in a protein kinase A-dependent pathway involving Rap1 and Raf (but not Ras) molecules.

Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase.

Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [35S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low Km for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [gamma-32P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.

Nucleolar Localization of Mouse Mammary Tumor Virus Proteins in T-Cell Lymphomas

To characterize novel proteins expressed in lymphoma cells, monoclonal antibodies (MAbs) were raised against variant S49 mouse lymphoma cells. Immunoperoxidase analysis with a specific MAb, named M-66, revealed nuclear localization with prominent staining in the nucleoli of both tumorigenic (T-63) cells and nontumorigenic, immunogenic (T-25-Adh) cells. Weak signals were also observed in the cytoplasm and plasma membrane of both cells. Western blot analysis with M-66 antibody revealed a 14-kDa protein in nuclear extracts of both T-25-Adh and T-63 cells. An additional nuclear 21-kDa protein was evident only in T-63 cells. M-66 identified several clones from a T-25-Adh cDNA expression library. These clones demonstrated extensive homology (∼95% identity throughout their length) to the mouse mammary tumor virus (MMTV) env and LTR regions. Extensive amino acid sequence homology (∼90% identity) between the clones and the env protein was observed. M-66 identified the 14-kDa protein in another MMTV bearing T-cell lymphoma, EL-4. Immunoperoxidase analysis of EL-4 cells with M-66 also revealed prominent nucleolar staining. MMTV-negative cells and MMTV-positive cells of nonlymphocytic origin were devoid of both 14- and 21-kDa proteins. Moreover, an anti-MMTV gp52 (env) antibody precipitated the 21-kDa protein in T-63 cells. We thus suggest that MMTV bearing T-cell lymphomas express nucleolar proteins translated from the env region of MMTV.

Delivery of cytotoxic drugs from carrier cells to tumour cells by apoptosis.

A new drug delivery approach, apoptotic-induced drug delivery (AIDD), is presented that is based on apoptosis as a mechanism to trigger delivery of drugs from carrier cells. It was investigated whether apoptotic drug-loaded carrier cells could deliver drugs to tumour cells by various mechanisms, including drug release through a more permeable apoptotic cell membrane, and by phagocytosis of drug-loaded apoptotic cells by tumour cells. The feasibility of this novel concept was evaluated in an in vitro carrier cell model that consisted of S49 mouse lymphoma cells that apoptose upon exposure to dexamethasone (DX). Membrane permeability was evaluated by measurement of release of a fluorescent dye (calcein-AM, C-AM) from C-AM-loaded S49 cells. Phagocytotsis of fluorescent PKH-26-labeled S49 cells was determined in co-culture studies with rat glioma (RG-2) cells using fluorescence microscopy and flow cytometry. Cytotoxicity of RG-2 cells due to temozolomide (TMZ)-loaded S49 cells was evaluated by a colony formation assay following co-culture of these cells for up to 8h. Calcein release from S49 cells was enhanced by approximately 30% at 48 h following treatment with DX compared to control S49 cells. Based on both flow cytometric and microscopic analyses, RG2 phagocytized apoptotic S49 cells to a four- to sevenfold greater extent than control S49 cells at co-incubation times from 4-48 h. The TMZ-loaded apoptotic S49 cells caused the largest degree of toxicity, about 50% cell kill, whereas TMZ-loaded control S49 caused 30% cell kill. The preliminary data suggest that AIDD should be further explored.

An experimental model for infiltration of malignant lymphoma to the eye and brain.

Currently there is no adequate experimental model available whereby the lethal infiltration of malignant lymphoma to the eye and CNS can be studied. Variant S49 mouse lymphoma cells that exhibit cell-cell adhesion properties (named Rev-2-T-6) were inoculated intraperitoneally into Balb/C mice at the ages of 6-60 days postnatal. Mice inoculated between days 6-11 postnatal developed signs of eye and CNS involvement with an apparent peak (58% of mice) at day 7. None of the mice inoculated beyond day 11 exhibited such signs. Histological analysis of these sites revealed tumorous infiltrates into a variety of structures in the orbit, intraocular tissues, along the optic nerve and in the brain. Additional analysis of the histopathological data, based on the structures demonstrating the highest frequency of lymphoma infiltration, suggests preferred routes of lymphoma entry to the brain and eye. Thus, entry to the brain can occur mainly through the choroid plexus and cranial nerves or cranial nerve ganglia. Entry to the eye may occur from the brain (along the optic nerve), and through hematogenous infiltration of orbital structures. No data were found that would support retrograde infiltration of the lymphoma from the eye to the brain. These findings present an experimental model for addressing the molecular mechanisms that govern homing of malignant lymphoma to the eye and brain, as well as the development of experimental therapeutic modalities for malignant lymphoma in these organs.

Variant mouse lymphoma cells with modified response to interferon demonstrate enhanced immunogenicity.

We have previously developed an experimental model for the xenogenization of malignant lymphoma. From highly tumorigenic S49 mouse lymphoma cells that proliferate in suspension culture (designated T-25), we selected variant clones that grew as an adherent monolayer (designated T-25-Adh) and were non-tumorigenic in syngeneic mice. Furthermore, priming of syngeneic hosts with T-25-Adh cells protected them against subsequent challenges with the tumorigenic T-25 cells. Several lines of evidence have indicated that antigens of an endogenous mouse mammary tumor virus (MMTV) are involved in the immunogenicity of T-25-Adh cells. Since interferon (IFN) is known to affect retroviral assembly and maturation on the cell membrane, we have studied the effects of IFN on endogenous MMTV-related structures, as well as on the immunogenicity of T-25-Adh cells. We observed that mouse alpha and beta interferons affect the morphogenesis of intracellular MMTV-related precursors in the immunogenic T-25-Adh cells, but not in tumorigenic T-25 cells. From T-25-Adh cells we selected variants that were either high responders or low responders to the above-mentioned interferon effect. The high-response variants were significantly more protective against tumorigenic T-25 cells than the low-response variants. Involvement of MMTV-related antigens in the immune response of the host to T-25-Adh cells was further suggested by immunoelectron-microscopical analysis, demonstrating that antisera from mice, immunized with T-25-Adh cells, interacted specifically with cell-surface MMTV budding particles. These findings indicate a novel method for xenogenization of lymphoma cells by IFN. Since endogenous retroviruses are present in all tissues of the mouse, this approach might be applicable to a wide variety of tumors.

Decreased transcript expression coincident with impaired glycosylation in the β2-adrenergic receptor gene does not result from differences in the primary sequence

Variants of the S49 mouse lymphoma cell line exhibit multiple lesions along the pathway of cyclic AMP generation in response to β 2-adrenergic stimulation. Two such variants, β p and β d, are characterized by decreased receptor binding and mRNA expression, 50% and 25% of wild-type receptor expression, respectively. The rate of β 2-adrenergic receptor synthesis was measured and found to be decreased in the βd cells vis-à-vis the rate in wild type cells. The molecular mass of the β 2-adrenergic receptor in the S49 wild-type, β p and β d variant cells was estimated by labeling the receptor with the photoaffinity probe [ 125I]iodocyanopindololdiazirine. Receptor size was found to be 67 000 and 47 000 Da in the wild-type and 60 000 and 42 000 in the two variant cells. This 6 kDa discrepancy in mass was abolished upon treatment of labeled cell extracts with N-glycosidase F, suggesting the possibility of either N-terminal truncation or altered glycosylation of the receptor in the variant cells. To distinguish between these possibilities, we sequenced the β 2-adrenergic receptor gene and two kilobases of the 5′-non-coding region. No differences were found in the coding region of the gene from wild-type, β p and β d S49 cells suggesting that both the diminished expression and the decreased size of β 2-adrenergic receptor in the β p and β d S49 variants are related to impaired glycosylation of the receptor. This hypothesis was substantiated by the reduced retention of the variant cells' β 2-adrenergic receptor on immobilized WGA. Furthermore, growth of the S49 cells in the presence of the α-mannosidase II inhibitor, swainsonine, preferentially impaired the ability of the receptors derived from the variant cells to bind to WGA. These results imply that altered expression and glycosylation of G-protein-linked receptors occur as a consequence of one or more mutations outside the receptor's open reading frame. ©1997 Elsevier Science B.V. All rights reserved.