Cell adhesiveness is related to tumorigenicity in malignant lymphoid cells

Mouse lymphoma cells (S49) that grow in suspension culture were selected for increased tumorigenicity through continuous passages in syngeneic BALB/c mice. Developing tumors were classified as high grade malignant lymphoma, small noncleaved type. Variants were selected from these tumorigenic cells that were able to grow as a monolayer attached to their substrate, resembling, in this respect, fibroblastoid cells. Whereas the tumorigenic suspension-growing parental cells were able to induce progressive tumors with an inoculum as low as 100 cells per mouse, the adherent cells were unable to develop as tumors even at an inoculum of 1 X 10(8) cells per mouse. In addition, mice inoculated once with live adherent cells were immunized against 1 X 10(7) suspension-growing cells. Involvement of an immune response in the rejection of tumorigenic S49 cells was suggested by (a) adoptive transfer experiments in which spleen cells from immunized mice protected naive mice and (b) the appearance of antibodies in the sera of immunized syngeneic mice that specifically recognized both adherent and suspension-growing S49 cells and detected differences in [35S]methionine-labeled antigens from these cells. Antibodies raised in rabbits against adherent cells recognized three proteins of 34,000, 61,000, and 72,000 apparent molecular weight in radiolabeled adherent cell extracts that are either absent or present in small amounts in extracts of suspension-growing tumorigenic S49 cells. These findings, taken together with our previous report (Hochman, J., A. Katz, E. Levy, and S. Eshel, 1981, Nature (Lond.), 290:248-249), suggest the S49 system as a novel system for studying growth control in malignant lymphoid cells.     

Isolation and characterization of interferon-resistant variants from S49 mouse lymphoma

S49 mouse lymphoma cells were found to be extremely sensitive to the antiproliferative activity of interferon. These characteristics were studied to select for IFN-resistant cell variants. Some 0.6% of the parental S49 cell population were resistant to the antiproliferative and cytotoxic activities of IFN. The resistant cells were cloned and analyzed for their responses to several of the activities of IFN, namely, inhibition of encephalomyocarditis (EMC) virus, murine leukemia virus (MuLV) replications, and the induction of (2'-5') oligoadenylate synthetase. Among the clones selected some were highly resistant while others demonstrated only partial responsiveness to IFN. S49 cells demonstrate tubular structures in the cytoplasm. These structures were previously reported to be antigenically related to mouse mammary tumor virus (MMTV). We report here that IFN treatment decreases the expression of these cytoplasmic viral structures as revealed by electron microscopy. To correlate this novel antiviral activity to the more established functions of IFN we utilized the above mentioned S49 IFN-resistant variants. The anti-MMTV activity of IFN correlated with the other effects of IFN in both the highly resistant and partially responsive S49 clones. Our findings indicate that a relatively high proportion of S49 cells vary in their response to IFN. The defect in the resistant cells appears to affect a primary response to IFN which is common to its diverse activities. Furthermore, the effect of IFN on MMTV-related structures involves the usual pathway of IFN action.     

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 α and β 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. Received: 6 June 1995 / Accepted: 13 March 1997     

Tubuloreticular structures in S49 cells. Relation to cAMP-dependent protein kinase

Tubuloreticular structures (TRS) occur spontaneously in S49 mouse lymphoma cells grown in suspension culture. These structures appear in approx. 20% of the cells in electron microscopical cross-sections. Cloning of S49 cells in semi-solid agarose reveals that TRS are potentially present in all S49 cells. An increase of 60% in the frequency of TRS was observed following exposure of S49 cells to 2 X 10(-4) db-cAMP. This increase was not observed in mutant S49 cells lacking cAMP-dependent protein kinase activity. The data suggest that one possibility for the regulation of TRS is through a pathway involving cAMP-dependent protein kinase. In addition, TRS also appear in tumors derived from S49 cells and therefore serve as an ultrastructural cytoplasmic marker for these cells, both in culture and in syngeneic hosts. We suggest the S49 cells as a model system for studying the regulation and function of tubuloreticular structures in vitro and in vivo in malignant lymphoid cells.     

Growth of hepatitis A virus in a mouse liver cell line

Hepatitis A virus (HAV) has been adapted to grow efficiently in primate and some nonprimate cell lines but not in cells of murine origin. To understand the inability of the virus to grow in mouse cells, we studied the replication of HAV in immortalized and nontransformed MMH-D3 mouse liver cells, which require growth factors and collagen to maintain their phenotype. HAV grew in MMH-D3 cells transfected with virion RNA but not in those infected with viral particles, indicating a cell entry block for HAV. However, MMH-D3 cells cultured under suboptimal conditions in the absence of growth factors acquired susceptibility to HAV infection. Serial passages of the virus in MMH-D3 cells under suboptimal growth conditions resulted in the selection of HAV variants that grew efficiently in MMH-D3 cells cultured under both optimal and suboptimal conditions. Nucleotide sequence analysis of the MMH-D3 cell-adapted HAV revealed that N1237D and D2132G substitutions were present in the capsid regions of six viral clones. These two mutations are most likely located on the surface of the virion and may play a role in the entry of HAV into the mouse liver cells. Our results demonstrate that mouse hepatocyte-like cells code for all factors required for the efficient growth of HAV in cell culture.     

Deoxyadenosine metabolism and cytotoxicity in cultured mouse T lymphoma cells: a model for immunodeficiency disease.

The inherited absence of either adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with severe immunological impairment. We have developed a cell culture model using a mouse T cell lymphoma to simulate ADA deficiency and to study the relationship between purine salvage enzymes and immune function. 2′-deoxyadenosine triphosphate (deoxyATP) levels have been shown to be greatly elevated in erythrocytes of immunodeficient, ADA-deficient patients, suggesting that deoxyadenosine is the potentially toxic substrate in ADA deficiency. Using a potent ADA inhibitor, we have demonstrated that deoxyadenosine is growth-inhibitory and cytotoxic to S49 cells, and that deoxyATP accumulates in these cells. Cell variants, unable to transport or phosphorylate deoxyadenosine, are much less sensitive to deoxyadenosine, indicating that intracellular phosphorylation of deoxyadenosine is required for the lethal effects.We have partially reversed the cytotoxic effects of deoxyadenosine with deoxycytidine in wild-type cells, but we cannot show any reversal in cell lines lacking deoxycytidine kinase. Adenosine (ado) kinase-deficient cells are extremely resistant to deoxyadenosine in the presence of deoxycytidine. This deoxycytidine reversal of deoxyadenosine toxicity is consistent with an inhibition of ribonucleotide reductase by deoxyATP, and we have shown that incubation of S49 cells with deoxyadenosine markedly reduces intracellular levels of deoxyCTP, deoxyGTP and TTP.Kinetics data in wild-type cells and in cell variants are consistent with the presence of two deoxyadenosine-phosphorylating activities — one associated with ado kinase and another associated with deoxycytidine kinase.The S49 cells appear to be a valid model for the simulation of ADA deficiency in cell culture, and from our results, we can suggest administration of deoxycytidine as a pharmacological regimen to circumvent the clinicopathologic symptoms in ADA deficiency.     

Interferon inhibition of the primary in vitro antibody response to a thymus-independent antigen

Partially purified and crude mouse L cell interferon preparations inhibited the in vitro plaque-forming cell (PFC) response of mouse C57B1/6 spleen cells to the T-cell independent lipopolysaccharide antigen of Escherichia coli 0127. PFC responses of 5-day cultures were inhibited approximately 70–90% by 100–200 NIH reference units of interferon/culture. A similar inhibitory effect was obtained with spleen cells from athymic (nude) mice homozygous for the nu/nu allele. Spleen cultures depleted of adherent cells were also inhibited in their anti-0127 PFC response by interferon. Interferon, then, appears capable of inhibiting the PFC response to E. coli 0127 via direct action on B cells. Heating experiments along with the use of interferon preparations of different specific activities suggest that the inhibition was due to the interferon in the preparations.     

Endotoxin-induced interferon-gamma production in culture cells derived from BCG-infected C3H/HeJ mice

The cultured cells prepared from the spleens and peritoneal exudate cells of the C3H/HeJ strain of mice produce very little or no interferon (IFN) by stimulation of bacterial lipopolysaccharide (LPS). However, the cells taken from LPS-non-responder C3H/HeJ mice which had been infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) prior to the experiment were capable of producing IFN in culture in the presence of LPS. The peritoneal exudate cells of BCG-primed C3H/HeJ mice were separated into adherent cell and nonadherent cell populations by their adhesiveness to plastic culture dishes. IFN production required the presence of both these cell populations in the same culture, and the IFN activities produced were mainly IFN-gamma. The cultures with nonadherent cells and fixed adherent cells still produced IFN, but the cell cultures reconstituted with the BCG-primed cell population and unprimed cell population produce little if any IFN-gamma. Moreover, when both of the populations were cultured in Marbrook culture vessels separated by a membrane filter, the cultures produced very little or no IFN-gamma. These results indicate that there is a mechanism of IFN-gamma induction by LPS which requires the direct contact between adherent cells and nonadherent cells without the participation of any soluble factor(s) from the adherent cells. The producer cells were mainly in the nonadherent cell population. Previous treatment of nonadherent cells with anti-Thy-1.2 antibody, anti-Lyt-1.1 antibody, anti-L3T4 antibody, or anti-asialo-GM1 antibody and complement diminished the ability of the cells for LPS-induced IFN production with the help of adherent cells. Therefore, it is concluded that both T cells (presumably L3T4+T cells) and asialo-GM1+ natural killer cells in the BCG-primed C3H/HeJ cell cultures produced IFN-gamma in the presence of LPS, and the production was supported by the function of macrophages.     

Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation

Bone marrow stroma contains a unique cell population, referred to as marrow stromal cells (MSCs), capable of differentiating along multiple mesenchymal cell lineages. A standard liquid culture system has been developed to isolate MSCs from whole marrow by their adherence to plastic wherein the cells grow as clonal populations derived from a single precursor termed the colony-forming-unit fibroblast (CFU-F). Using this liquid culture system, we demonstrate that the relative abundance of MSCs in the bone marrow of five commonly used inbred strains of mice varies as much as 10-fold, and that the cells also exhibit markedly disparate levels of alkaline phosphatase expression, an early marker of osteoblast differentiation. For each strain examined, the method of isolating MSCs by plastic adherence yields a heterogeneous cell population. These plastic adherent cells also exhibit widely varying growth kinetics between the different strains. Importantly, of three inbred strains commonly used to prepare transgenic mice that we examined, only cells derived from FVB/N marrow readily expand in culture. Further analysis of cultures derived from FVB/N marrow showed that most plastic adherent cells express CD11b and CD45, epitopes of lymphohematopoietic cells. The later consists of both pre-B-cell progenitors, granulocytic and monocytic precursors, and macrophages. However, a subpopulation of the MSCs appear to represent bona fide mesenchymal progenitors, as cells can be induced to differentiate into osteoblasts and adipocytes after exposure to dexamethasone and into myoblasts after exposure to amphotericin B. Our results point to significant strain differences in the properties of MSCs and indicate that standard methods cannot be applied to murine bone marrow to isolate relatively pure populations of MSCs.     

Isolation and characterization of an archaebacterial viruslike particle from Methanococcus voltae A3.

Small amounts of a 23-kilobase covalently closed circular DNA molecule were isolated from unwashed cells of Methanococcus voltae A3. Further investigation indicated the presence of greater quantities of the circular DNA in the culture supernatant, complexed with protein in a manner rendering the DNA resistant to DNase. Electron-microscopic examination of supernatant material revealed the presence of particles which morphologically resemble virus. Phenol extraction of viruslike particle preparations resulted in the recovery of DNase-sensitive open-circular DNA molecules. As many as 30 viruslike particles per cell were recovered from some cultures. Hybridization data clearly indicated the presence of a chromosomally integrated copy of the viruslike particle DNA. Although M. voltae PS was not observed to produce viruslike particles, DNA homologous to the viruslike particle DNA was detected in its chromosome. A mutant of M. voltae A3 was isolated which produced no particles; its DNA was deleted for 80% of the integrated viruslike particle DNA. Despite any similarities to lysogenic bacteriophages of eubacteria, neither infectivity nor inducibility of the viruslike particles could be demonstrated.     

Unusual lysosomes in aortic smooth muscle cells: presence in living and rapidly frozen cells

Unusual tubular structures have been observed in rat aortic smooth muscle cells (SMC) grown in culture. These tubular structures have several characteristics that strongly suggest that they are lysosomes: they are bounded by a single membrane bilayer, contain densely staining material, and acid phosphatase activity. Furthermore, these structures are present in living cells, as demonstrated by their ability to accumulate the membrane-impermeable fluorescent dye lucifer yellow CH. In ultrastructural preparations they are best seen in samples that are cryofixed by rapid freezing and then freeze-substituted in osmium-acetone solutions. Conventional chemical fixation did not appear to preserve these structures to as great an extent as did rapid freezing. Comparison of SMC in vitro to the same cells in situ revealed differences in lysosome number as well as morphological appearance. Thus, the culturing of rat SMC leads to the formation of unusual tubular lysosomes whose ultrastructural appearance is particularly sensitive to the methods employed for examination.     

Monocyte and NK cell cytotoxic activity in human adherent cell preparations: discriminating effects of interferon and lactoferrin

The comparative cytotoxic specificities of freshly isolated human adherent and nonadherent blood mononuclear cells were examined against seven established target cell lines in 4 and 18 hr chromium release assays. The relative sensitivity of each target cell line to the cytotoxic effects of both adherent and nonadherent effector cells in cultures was identical. Moreover, the relative enhancing effects of interferon on cytotoxicity by both effector cell types were also identical. These adherent cell preparations were contaminated with up to 6% NK cells, as demonstrated by OKM1 staining and flow microfluorometry. These NK cells were loosely adherent and could be removed by vigorous wash procedures. The remaining tightly adherent monocytes also had the capacity to kill K562 cells and Chang cells, but these cytotoxic effects could not be increased by interferon. Enhancement by lactoferrin, however, was consistently observed. Treatment of mononuclear cells with Leu-lla, a monoclonal antibody that reacts with all NK cells, also abolished the enhancing effects of interferon, but not lactoferrin. These studies suggest that caution must be exercised in attributing all cytotoxic activities in adherent cell preparations to monocytes, and that lactoferrin and interferon can be used as functional probes to detect two distinct blood mononuclear cell subsets with natural cytotoxicity.     

Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation

Ultraviolet irradiation (UV) of cadmium-sensitive S49 mouse cells induces a large increase in cadmium-resistant variants. About 30%-40% of these variants make metallothionein (MT)-I mRNA while S49 cells do not. S49 cells contain two copies of the MT-I gene; both alleles are heavily methylated but can be conveniently distinguished by the methylation status of a single Hpa II site. In lines expressing MT-I, one allele becomes completely demethylated at all methylation-sensitive restriction sites examined over at least a 2.5 kb region spanning the MT-I gene. Activation of a quiescent gene by UV has implications for understanding the initiation of carcinogenesis.     

Fetal Kidney Cells Can Ameliorate Ischemic Acute Renal Failure in Rats through Their Anti-Inflammatory,Anti-Apoptotic and Anti-Oxidative Effects

Fetal kidney cells may contain multiple populations of kidney stem cells and thus appear to be a suitable cellular therapy for the treatment of acute renal failure (ARF) but their biological characteristics and therapeutic potential have not been adequately explored. We have culture expanded fetal kidney cells derived from rat fetal kidneys, characterized them and evaluated their therapeutic effect in an ischemia reperfusion (IR) induced rat model of ARF. The fetal kidney cells grew in culture as adherent spindle shaped/polygonal cells and expressed CD29, CD44, CD73, CD90, CD105, CD24 and CD133 markers. Administration of PKH26 labeled fetal kidney cells in ARF rats resulted in a significant decrease in the levels of blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin and decreased tubular necrosis in the kidney tissues (p<0.05 for all). The injected fetal kidney cells were observed to engraft around injured tubular cells, and there was increased proliferation and decreased apoptosis of tubular cells in the kidneys (p<0.05 for both). In addition, the kidney tissues of ARF rats treated with fetal kidney cells had a higher gene expression of renotropic growth factors (VEGF-A, IGF-1, BMP-7 and bFGF) and anti-inflammatory cytokine (IL10); up regulation of anti-oxidative markers (HO-1 and NQO-1); and a lower Bax/Bcl2 ratio as compared to saline treated rats (p<0.05 for all). Our data shows that culture expanded fetal kidney cells express mesenchymal and renal progenitor markers, and ameliorate ischemic ARF predominantly by their anti-apoptotic, anti-inflammatory and anti-oxidative effects.     

In vitro differentiation of mouse embryo chondrocytes: requirement for ascorbic acid.

Chondrocytes enzymatically dissociated from 13-day-old mouse embryo tibia grow in monolayer culture with a fibroblast-like phenotype and express high levels of type I collagen. Chondrogenesis can be induced by transferring the adherent cells in suspension culture and maintaining them in the constant presence of mouse embryo extract. Round shaping of the cells and formation of multicellular aggregates rapidly follow the passage in anchorage-independent conditions. Cell differentiation is evidenced by a marked decrease in the level of type I collagen and by the induction of type II collagen which accumulates when ascorbic acid is included in the culture medium. The addition of the vitamin also triggers the aggregated chondrocytes to organize their extracellular matrix giving rise to a structure closely resembling the in vivo developing cartilage.     

Temporal distribution of CDK4, CDK6, D-type cyclins,and p27 in developing mouse oocytes

Various molecular interactions not operating in other cell types are most likely required for mammalian oocytes to develop into fully competent eggs. This study seeks to initiate analyses of the potential oocyte-specific functions of regulators of G1/S progression-CDK4, CDK6, D-type cyclins, and p27-by first determining their expression patterns in growing and maturing mouse oocytes and in mouse embryos early after fertilization. Western blot and immunofluorescence analyses on isolated oocytes were employed to evaluate both their levels and their localization. The data show that 1). mouse oocytes contain significant amounts of all studied regulators; 2). their amounts and localization undergo dramatic changes as the oocytes grow, meiotically mature, and transit into embryogenesis; and 3). some regulators (CDK4, CDK6, cyclin D2, and p27) appear in unusual, most likely posttranslationally modified, forms. These data distinguish G1/S regulators as the potential players in molecular processes that are important for oocytes to function normally.     

Polyoma virus middle-T-transformed PECAM-1 deficient mouse brain endothelial cells proliferate rapidly in culture and form hemangiomas in mice

Wild-type mouse brain endothelial (bEND) cells transformed with the polyoma virus middle-T proliferate rapidly in culture and form hemangiomas in mice. These cells express high levels of platelet/endothelial cell adhesion molecule-1 (PECAM-1), a molecule shown to be important during hemangioma formation. In this study, we have examined the ability of polyoma virus middle-T-transformed mouse bEND cells prepared from PECAM-1-/- mice to proliferate in culture and form hemangiomas in mice. We show that these cells express a number of endothelial cell markers and share a similar morphology with PECAM-1+/+ bEND cells. PECAM-1-/- bEND cells exhibit a limited ability to form tubes in Matrigel and rapidly form hemangioma when injected into nude mice, very similar to PECAM-1+/+ bEND cells. These cells, however, have increased proliferation, slower migration, altered endothelial cell adhesion molecule expression, and are less adherent when compared to PECAM-1+/+ bEND cells. Therefore, lack of PECAM-1 expression impacts polyoma middle-T-transformed endothelial cell proliferative, adhesive, and migratory properties without impacting their ability to rapidly form hemangiomas in mice or poorly organize to capillary-like structures in Matrigel.     

Hemoglobin and ferritin synthesis in erythroid cells in prolonged marrow cell cultures

A method is described which maintains viable erythroid cells in tissue culture for periods from nine to twenty days. These cells appear predominantly as small round cells with scanty cytoplasm. They synthesize both heme and globin and are relatively more numerous free in suspension than in the adherent monolayer. Ferritin isomorph may serve as a convenient marker in tissue culture of cells of erythroid origin, suggesting that such cells may persist despite a completely transformed appearance and a loss of the ability to produce hemoglobin.     

In vitro colony assay for a new class of megakaryocyte precursor: colony-forming unit megakaryocyte (CFU-M).

A plasma culture system has been used successfully to grow and quantitate megakaryocyte colonies from mouse bone marrow following their staining for acetylcholinesterase activity in situ. Colonies averaging about six acetylcholinesterase-positive cells appear with a peak incidence after 4 days in culture with a plating efficiency of one colony formed for every 10(4) nucleated cells plated.