Cytogenesis of murine mammary tumors in BALB/cfC3H and BALB/cfRIII strains

Biological and morphological differences in the mammary tumors of BALB/cfC3H and BALB/cfRIII mice are due to differences in the causative viruses. The C3H and RIII variants of the murine mammary tumor virus (MuMTV) might give origin to different mammary tumors by transforming different types of cell, i.e. epithelial or myoepithelial cells. The nature (epithelial or myoepithelial) of the neoplastic cells has been investigated by demonstrating their plasma membrane ATPase activities. We found that in normal murine mammary gland both epithelium and myoepithelium have Mg++ dependent ATPase activity, while the myoepithelium shows in addition an Na+K+ dependent ATPase activity. It is suggested that the results obtained exclude the participation of myoepithelium to the neoplastic growth and we ascribe the differences in mammary tumors of the two strains of mice to differences in the mechanisms of action of the virus variants.     

Murine Mammary Tumor Virus Expression During Mammary Tumorigenesis in BALB/c Mice

Steady-state levels of murine mammary tumor virus (MuMTV) RNA were quantitated during mammary tumorigenesis in BALB/c mice by molecular hybridization with a representative MuMTV complementary DNA (cDNA) probe. Hyperplastic alveolar nodule (HAN) lines are preneoplastic mammary lesions that were induced in BALB/c mice by hormones alone or in combination with 7,12-dimethylbenz(a)anthracene and give rise to mammary tumors. The hormone-induced HAN lines D1 and D2 contained detectable amounts of hybridizable MuMTV sequences. MuMTV RNA sequences were also observed in five of the six transplanted BALB/c mammary tumors that were examined. Similar levels of hybridizable MuMTV RNA were observed between the D1 or D2 HAN line and mammary tumors derived from each HAN line. The D2 HAN line as well as D2, C4, and CD8 mammary tumors accumulated RNA that was apparently homologous to most of the MuMTV genome. Thermal denaturation of hybrids indicated extensive sequence homology between the MuMTV cDNA and hybridizable RNA in the BALB/c HAN lines and mammary tumors. A low level of type C viral RNA was observed in the BALB/c HAN lines and most mammary tumors by molecular hybridization with a cDNA to Moloney murine leukemia virus. These data demonstrate that MuMTV sequences are frequently expressed in hormone-induced BALB/c HAN lines and mammary tumors derived from HAN lines or ductal hyperplasias induced in BALB/c mice by hormones and/or a chemical carcinogen. The transition from the preneoplastic to the neoplastic state in BALB/c mice does not appear to be due to a change in the steady-state levels of MuMTV RNA since the hormone-induced HAN lines and mammary tumors had similar levels of hybridizable MuMTV RNA.     

Ultrastructural localizations of adenosine triphosphatase activity in resting mammary gland.

Adenosine triphosphatase (ATPase) activity was localized at an ultrastructural level in the resting mammary glands of female BALB/c mice. A Mg++ dependent ATPase was localized in the plasma membranes of both the epithelial and myoepithelial cells of the mammary tubules. A second type of ATPase activity that was not Mg++-dependent but that was Na+ and K+ dependent was localized primarily in the plasma membranes of the myoepithelial cells. Preincubation with either ouabain or N-ethylmaleimide decreased the quantity of reaction product, indicating that both types of ATPase activity were sensitive to these inhibitors. Control media, containing adenosine triphosphate and Pb(NO3)2 without cations, demonstrated that the amount of nonezymatic hydrolysis was negligible. These differences in the cationic requirements for plasma membrane ATPase activity can be used to distinguish histochemically the epithelial from myoepithelial cells in mammary tissue.     

High-resolution 2-dimensional protein mapping of “diploid” and “aneuploid” mammary adenocarcinomas

Summary Two-dimensional-electrophoretic analysis has been applied to non-neoplastic mammary epithelium from eight healthy women, tumor tissue from eight diploid mammary adenocarcinomas, and tumor tissue from eight aneuploid mammary adenocarcinomas. Compared with non-neoplastic mammary epithelium, a slight numerical net increase of the protein spots was detected in diploid tumors and a marked increase in aneuploid tumors. Two prominent spots were present in all 16 malignant tissues examined and absent in all eight non-neoplastic tissues (silver staining method).The results suggest that a difference in the composition of cellular proteins exists both between non-neoplastic mammary cells and malignant tumor cells, and between diploid and aneuploid tumors.In honour of Prof. P. von Duijn     

Lithium transport across isolated frog skin epithelium

Summary Transepithelial Li⁺ influx was studied in the isolated epithelium from abdominal skin ofRana catesbeiana. With Na⁺-Ringer's as inside medium and Li⁺-Ringer's as outside medium, the Li⁺ influx across the epithelium was 15.6 A/cm². This influx was considerably reduced by removal of either Na⁺ or K⁺ from the inside bath or by the addition of ouabain or amiloride. Epithelial K⁺ or Na⁺ concentration was respectively lower in epithelia bathed in K⁺-free Ringer's or Na⁺-free Ringer's. In conditions of negligible Na⁺ transport, a 20mm Li⁺ gradient (outin) produced across the short-circuited epithelium a Li⁺ influx of 11.8 A/cm² and a mean short-circuit current of 10.2 A/cm². The same Li⁺ gradient in the opposite direction produced a Li⁺ outflux of only 1.9 A/cm². With equal Li⁺ concentration (10.3 and 20.6mm) on both sides of the epithelium, plus Na⁺ in the inside solution only, a stable Li⁺-dependent short-circuit current was observed. Net Li⁺ movement (outin) was also indirectly determined in the presence of an opposing Li⁺ gradient. Although Li⁺ does not substitute for Na⁺ as an activator of the (Na⁺+K⁺)-ATPase from frog skin epithelium, Li⁺ influx appears to be related to Na⁺–K⁺ pump activity. It is proposed that the permeability of the outer barrier to Na⁺ and Li⁺ is regulated by the electrical gradient produced by electrogenic Na⁺–K⁺ pumps located in the membrane of the deeper epithelial cells.     

Evolutionary temperature adaptation of fish sarcoplasmic reticulum

Summary Sarcoplasmic reticulum has been isolated from the white muscle of 15 species of teleost fish adapted to diverse thermal environments. Evidence has been obtained that the Ca²⁺-dependent ATPase of fish sarcoplasmic reticulum has undergone evolutionary modification for function at different temperatures. Compared with tropical fish, cold adapted species have higher rates of Ca²⁺ transport and Ca²⁺-ATPase activities at low temperatures. Most species have linear Arrhenius plots over the temperature range 0–30°C. Activation enthalpies (H ) of the ATPase ranged from 53–190 kJ mol^–1 and were positively correlated with environment temperature. Activation entropy (S ) varied from negative values in cold adapted species to positive values in tropical fish.In contrast to the Ca²⁺-ATPase, the basal ATPase of fish sarcoplasmic reticulum showed no relationship between either ATPase activity or thermodynamic activation parameters and environmental temperature.Only the Ca²⁺-dependent ATPase is coupled to Ca²⁺ transport. The percentage of total ATPase activity which is Ca²⁺ activated is higher at low temperatures in cold than in warm adapted species. For example, ratios of Ca²⁺-dependent/total ATPase at 2°C varied from 80–98% in Arctic, Antarctic and North Sea species to only 2–50% in various tropical fish. Above 20°C, similar ratios in the range 80–98% were obtained for all species. The nature of the basal ATPase and mechanisms of temperature adaptation of fish sarcoplasmic reticulum are discussed.Abbreviations ET environmental temperature - EGTA ethylene glycol-bis (-aminolethyl ether)-N, N-tetraacetic acid - HEPES N-2-hydroxylpiperazine-N-2-ethanesulfonic acid - SR sarcoplasmic reticulum     

Stromal Cell-Derived Factor-1/CXCL12 Contributes to MMTV-Wnt1 Tumor Growth Involving Gr1+CD11b+ Cells

Background

Histological examinations of MMTV-Wnt1 tumors reveal drastic differences in the tumor vasculature when compared to MMTV-Her2 tumors. However, these differences have not been formally described, nor have any angiogenic factors been implicated to be involved in the Wnt1 tumors.

Methodology/Principal Findings

Here, we show that MMTV-Wnt1 tumors were more vascularized than MMTV-Her2 tumors, and this correlated with significantly higher expression of a CXC chemokine, stromal cell-derived factor-1 (SDF1/CXCL12) but not with VEGFA. Isolation of various cell types from Wnt1 tumors revealed that SDF1 was produced by both tumor myoepithelial cells and stromal cells, whereas Her2 tumors lacked myoepithelial cells and contained significantly less stroma. The growth of Wnt1 tumors, but not Her2 tumors, was inhibited by a neutralizing antibody to SDF1, but not by neutralization of VEGFA. Anti-SDF1 treatment decreased the proportion of infiltrating Gr1⁺ myeloid cells in the Wnt1 tumors, which correlated with a decrease in the percentage of endothelial cells. The involvement of Gr1⁺ cells was evident from the retardation of Wnt1 tumor growth following in vivo depletion of these cells with an anti-Gr1-specific antibody. This degree of inhibition on Wnt1 tumor growth was comparable, but not additive, to the effect observed with anti-SDF1, indicative of overlapping mechanisms of inhibition. In contrast, Her2 tumors were not affected by the depletion of Gr1⁺ cells.

Conclusions/Significance

We demonstrated that SDF1 is important for Wnt1, but not for HER2, in inducing murine mammary tumor and the role of SDF1 in tumorigenesis involves Gr1⁺ myeloid cells to facilitate growth and/or angiogenesis.     

PC12 variants deficient in norepinephrine transporter mRNA have wild type activities of several other related transporters

Wild type PC12 pheochromocytoma cells express a Na⁺-dependent norepinephrine transporter that operates in the uptake of catecholamines. In addition to the previously described Na⁺-dependent system A for the uptake of -amino-isobutyric acid and system Gly for glycine, we have identified two other Na⁺-dependent transporter systems for amino acid uptake in these cells: 1) system for -alanine and taurine; and 2) a system for creatine. Uptake of -amino-isobutyric acid, glycine, -alanine, and creatine is not affected in some PC12 variants that were previously shown to be deficient in catecholamine uptake and to have decreased levels of norepinephrine transporter mRNA. We have isolated two PC12 cDNA clones that are essentially identical in sequence to recently reported cDNAs for rat brain taurine and creatine transporters, respectively, and a third cDNA that appears to code for a novel transporter. mRNAs for these three transporters are present at wild type levels in those variants that express no or little norepinephrine transporter mRNA. These results support the notion that the expression of catecholamine reuptake transporters may be particularly susceptible to down-regulation.     

Gene conversion accounts for pilin structural changes and for reversible piliation “phase” changes in gonococci

Pilus⁺ wild-type gonococci (Gc) frequently display gene conversion of their expressed complete pilin gene (CPG); a copy of DNA derived from one of the Gc genome's multiple silent partial pilin genes (PPG) is recombinationally-inserted into the CPG's central and 3 portions with formation of a new, chimeric CPG. Expression of that new CPG leads to either 1) retention of pilus⁺ phenotype but change in pilin primary structure/antigenicity, or 2) phase change to pilus^– phenotype capable of reverting. This study utilizes pilus revertants of P^– rp±Gc and P⁺ colony morphotye variants spawned by P⁺⁺ Gc to examine pilin gene conversion in strain MS11_mk Gc in greater detail. Each revertant's and variant's expressed pilin gene's sequence (as pilin mRNA) was defined to learn whether their differences are due to gene conversion by different PPGs, or by varying stretches from the same PPG, or both. Gene conversion by PPG pilS1 copy 2 has been documented in Gc recovered from a human voluteer's urethra previously inoculated with pilus Gc (strain MS11). The pilus⁺ Gc isolated expressed structurally/antigenically distinct pilins.     

Effects of orally administered retinol on natural killer cell activity in wild type BALB/c and congenitally athymic BALB/c mice

Summary Retinoids have been shown to inhibit the growth and development of neoplastic cells in many systems. One mechanism of action may be through activation of the immune system, specifically natural killer (NK) cell activity. The effect of retinol on NK cell cytotoxicity was examined in three groups of mice: BALB/c (wild-type), BALB/c nu/nu (athymic), and BALB/c nu/nu previously injected with human tumor cells. In untreated mice, NK activity was highest in athymic mice without tumors and lowest in wild-type mice, although serum and liver retinol concentrations were identical in all three groups. In mice fed graded, nontoxic doses of retinol daily for 3 weeks, serum retinol levels in all three groups exhibited a sharp peak and decline following daily bolus retinol administration. Retinol stores in the livers showed a dose-dependent increase in all treated animals. However, NK cell activity, differed for each group. Athymic mice without tumors exhibited no change in NK activity as a result of retinol treatment. Athymic mice with tumors had NK levels that tended to increase with increasing retinol doses, but these changes were not statistically significant. Wild-type mice, on the other hand, demonstrated significantly higher NK levels after treatment with retinol doses of 300 and 600 g/day. In subsequent time course experiments, there was a peak in NK activity 1 h following bolus retinol administration similar to the peak seen in serum retinol concentrations, suggesting either an acute activation or recruitment of cytotoxic cells. Retinol thus appears to increase NK activity in wild-type BALB/c mice, and this activity may be an important component of its antineoplastic activity.This investigation was supported by Biomedical Research Support Grant RRO 5424, the Veterans Administration, and PHS Grant number CA 33589-01A2, awarded by the National Cancer Institute, DHHSThis work was done in partial fulfillment of a Ph. D. thesis by L. Fraker in the Department of Pathology, Vanderbilt University School of Medicine     

Evidence of voltage-induced channel opening in Na/K ATPase of human erythrocyte membrane

Summary Previous studies have shown that human erythrocytes when subjected to a high voltage pulsation, in the microsecond time range, lysed in an isotonic medium. The hemolysis was the result of the colloid osmotic swelling, which, in turn, was caused by the voltage perforation of the red cell membranes. In this work we demonstrate that in a low ionic medium at least 35% of the pores was related to the opening of Na⁺/K⁺ ATPase channels. The membrane conductance generated by the externally applied electric field could be partially blocked by a specific inhibitor, ouabain, or by a specific cross-linking reagent, Cu⁺⁺-phenanthroline, of the ATPase. The effect of ouabain was saturable and had a mid-point of saturation at 0.15 m. This value agrees with the physiological inhibition constant of the drug. K⁺ ion in the external medium suppressed the effect of ouabain, as has also been demonstrated in physiological studies. Experiment presented in this communication also suggests that the Na⁺/K⁺ ATPase was not perforable in a high ionic medium, and that a large fraction of the voltage-induced pores occurred at as yet unidentified sites.     

Antitumor effect of combination of murine recombinant interferon β, murine recombinant interferon γ and human recombinant interleukin-2 in MethA-bearing mice

Summary We have previously reported that the combination of murine recombinant interferon (Mu-rIFN) with murine recombinant interferon (Mu-rIFN) provided greater inhibition of tumor growth than did each one alone in MethA-bearing mice. In the present study the effect of addition of human recombinant interleukin-2 (Hu-rIL-2) to the combination of Mu-rIFN with Mu-rIFN on tumor growth in BALB/c mice bearing syngeneic MethA fibrosarcoma was examined. Low doses of Hu-rIL-2 (5 × 10³ U or 5 × 10⁴ U at 3-day intervals) showed no antitumor activity, while a high dose of Hu-rIL-2 (5 × 10⁵ U) showed profound growth inhibition. The administration of IL-2 (ranging between 5 × 10³ U and 5 × 10⁵ U) in addition to the combination of IFN and IFN showed more augmented antitumor effects in a dose-dependent manner. Furthermore, the simultaneous administration of IL-2, IFN and IFN had more effective therapeutic activity, compared with the sequential administration of interferons and IL-2. These findings indicated that IL-2 in combination with IFN and was effective for cancer treatment.     

Transforming growth factor-β1 immobilises dendritic cells within skin tumours and facilitates tumour escape from the immune system

Human skin tumours often regress spontaneously due to immune rejection. Murine skin tumours model this behaviour; some regress and others progress in syngeneic immunocompetent hosts. Previous studies have shown that progressor but not regressor skin tumours inhibit dendritic cell (DC) migration from the tumour to draining lymph nodes, and transforming growth factor-₁ (TGF-₁) has been identified as a responsible factor. To determine whether increased production of TGF-₁ in the absence of other differences inhibits DC migration from the tumour and enables it to evade immune destruction, a murine regressor squamous cell carcinoma clone was transfected with the gene for TGF-₁. This enhanced growth in vitro and in vivo, causing it to become a progressor. TGF-₁ transfection reduced the number of infiltrating DCs by about 25%. Quantitation of CD11c⁺ E-cadherin⁺ (epidermally derived) DCs in lymph nodes determined that TGF-₁ reduced the number of DCs that migrated from the tumour to undetectable levels. This was supported by showing that TGF-₁ reduced DC migration from cultured tumour explants by greater than tenfold. TGF-₁ transfection also reduced the number of infiltrating CD4 and CD8 T cells. Thus, TGF-₁ production by skin tumours is sufficient to immobilise DCs within the tumour, preventing their migration to lymph nodes. This reduces the number of T cells that infiltrate the tumour, preventing regression. Thus, TGF-₁ is a key regulator of whether skin tumours regress or progress.     

Vacuolar H+-translocating ATPases from plants: Structure,function, and isoforms

The vacuolar H⁺-translocating ATPase (V-type ATPase) plays a central role in the growth and development of plant cells. In a mature cell, the vacuole is the largest intracellular compartment, occupying about 90% of the cell volume. The proton electrochemical gradient (acid inside) formed by the vacuolar ATPase provides the primary driving force for the transport of numerous ions and metabolites against their electrochemical gradients. The uptake and release of solutes across the vacuolar membrane is fundamental to many cellular processes, such as osmoregulation, signal transduction, and metabolic regulation. Vacuolar ATPases may also reside on endomembranes, such as Golgi and coated vesicles, and thus may participate in intracellular membrane traffic, sorting, and secretion.Plant vacuolar ATPases are large complexes (400–650 kDa) composed of 7–10 different subunits. The peripheral sector of 5–6 subunits includes the nucleotide-binding catalytic and regulatory subunits of 70 and 60 kDa, respectively. Six copies of the 16-kDa proteolipid together with 1–3 other subunits make up the integral sector that forms the H⁺ conducting pathway. Isoforms of plant vacuolar ATPases are suggested by the variations in subunit composition observed among and within plant species, and by the presence of a small multigene family encoding the 16-kDa and 70-kDa subunits. Multiple genes may encode isoforms with specific properties required to serve the diverse functions of vacuoles and endomembrane compartments.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - CAM Crassulacean acid metabolism - Nbd-Cl 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole - Bz-ATP 3-O-(4-benzoyl)benzolyadenosine 5-triphosphate - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - NEM N-ethylmaleimide - IP₃ inositol-1,4,5-triphosphate - H⁺-PPase H⁺-translocating pyrophosphatase - V-type vacuolar-type - P-type phosphorylated intermediate- or plasma membrane-type - F-type F₁F_o-type - V-ATPase vacuolar-type H⁺-ATPase     

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models. Download video file.^{(81M, mov)}     

Diversity,metabolic types and δ13C carbon isotope ratios in the grass flora of Namibia in relation to growth form,precipitation and habitat conditions

The grass flora of Namibia (374 species in 110 genera) shows surprisingly little variation in ¹³C values along a rainfall gradient (50–600 mm) and in different habitat conditions. However, there are significant differences in the ¹³C values between the metabolic types of the C4 photosynthetic pathway. NADP-ME-type C4 species exhibit the highest ¹³C values (–11.7 ) and occur mainly in regions with high rainfall. NAD-ME-type C4 species have significantly lower ¹³C values (–13.4 ) and dominate in the most arid part of the precipitation regime. PCK-type C4 species play an intermediate role (–12.5 ) and reach a maximum abundance in areas of intermediate precipitation. This pattern is also evident in genera containing species of different metabolic types. Within the same genus NAD species reach more negative ¹³C values than PCK species and ¹³C values decreased with rainfall. Also in Aristida, with NADP-ME-type photosynthesis, ¹³C values decreased from –11 in the inland region (600 mm precipitation) to –15 near the coast (150 mm precipitation), which is a change in discrimination which is otherwise associated by a change in metabolism. The exceptional C3 species Eragrostis walteri and Panicum heterostachyum are coastal species experiencing 50 mm precipitation only. Many of the rare species and monotypic genera grow in moist habitats rather than in the desert, and they are not different in their carbon isotope ratios from the more common flora. The role of species diversity with respect to habitat occupation and carbon metabolism is discussed.     

Ca2+ pumping ATPase of cardiac sarcolemma is insensitive to membrane potential produced by K+ and Cl− gradients but requires a source of counter-transportable H+

Summary The sensitivity of the Ca²⁺ pumping ATPase of bovine cardiac sarcolemma (SL) to changes in membrane potential was studied in a preparation of sealed SL vesicles. Membrane potential was imposed by preincubating the vesicles in media of defined ion composition (K⁺, Cl^–, choline⁺ and gluconate^–) and diluting into media of differing ion composition. The durations of the ion gradients and relative ion permeabilities were determined in separate experiments by the dependence of the half time for net K⁺ (or choline⁺) movement coupled with these anions (Cl^– or gluconate^–), registered by the fluorescence of 1-anilino-8-naphthalene sulfonate (Chiu, V.C.K., Jaumes. D.H. 1980.J. Membrane Biol. 56:203–218). Relative permeabilities were: 1.0, K⁺, 10.0, 1 m valinomycin-K⁺; 4.0, Cl^–, 0.66, choline⁺; 0.38, gluconate^–. Durations of the gradients ranged between 17 sec (KCl, valinomycin) to 195 sec (K⁺-gluconate^–). In separate experiments. active Ca²⁺ uptake was monitored using chlorotetracycline (CTC) fluorescence, a technique validated by 45-Ca²⁺ measureaments (Dixon, D., Brandt, N., Haynes, D.H. 1984.J. Biol. Chem. 259:13737–13741). Active Ca²⁺ uptake was initiated in the presence of monovalent ion gradients. The values of the membrane potentials (E _m ) imposed by the monovalent ion gradients were calculated using the ion concentrations, their relative permeabilities and the Goldman-Hodgkin-Katz equation. No effect of membrane potential on transport rate was observed (4%, for 5–7%sd) for imposed potentials as extreme as +71 and –67 mV. Formal analysis shows that the above observations are not compatible with models in which the Ca²⁺ pumping ATPase functions in an electrogenic or charge-uncompensated fashion. Further experimentation showed that the pump rate is slowed when uptake is measured at less-than-adequate concentrations of buffer (5vs. 25mm HEPES/Tris). This, together with further control experiments using nigericin and FCCP, gave evidence that the pump requires a source of counter-transportable H⁺ in the vesicle lumen. The above experimentation also underlines the need for control of internal pH to obviate erroneous interpretation of ion perturbation experiments. The results are compared with results obtained with the Ca²⁺ ATPase pump of skeletal sarcoplasmic reticulum.     

Immunotherapy in a spontaneously developed murine mammary carcinoma with syngeneic monoclonal antibody

Summary A new spontaneously arisen murine breast tumor, designated JC, has been established in immunocompetent BALB/c mice. Upon reestablishment of tumor in vitro and in vivo, the epithelial murine tumor cells retained their original papillary adenocarcinoma morphology. Various immunotherapy protocols have been performed in previously implanted and progressively growing JC tumor in syngeneic hosts with a murine monoclonal antibody (McAb), F36/22 (IgG3). Affinity of McAb binding to JC tumor cells was determined to be 6.1×10⁷ L/M. Quantitatively 1.2×10⁵ molecules of McAb bound to a JC tumor cell. Immunotherapeutic effectiveness in vivo on a tumor mass after its establishment is a major feature of this experimental tumor model. When four sequential administration of McAb, i.p., at a dose of 400 g 4 days apart were used, McAb-treated animals showed statistically significant tumor regression and longer survival than those of control animals treated with an irrelevant McAb of the same isotype. Two temporal phases of tumoricidal activity were observed as measured by tumor volume reduction. The first phase of tumoricidal response (tumor regression) was detected within days upon the first administration of McAb. A distinct second phase followed within 3–5 weeks after the last McAb administration, which resulted in tumor necrosis even in large tumors. Histological examinations revealed heavy infiltration of inflammatory cells at the beginning of the second phase. Similar tumor regression was also obtained from animals treated with a single dose (400 g) of McAb followed by injections of McAb with complete and incomplete adjuvant, respectively. These results demonstrate that this syngeneic murine mammary tumor can serve as a potential preclinical model for investigation of parameters and mechanisms associated with McAb immunotherapy.     

Mammary tumor modifiers in BALB/cJ mice heterozygous for p53

BALB/c mice are predisposed to developing spontaneous mammary tumors, which are further increased in a p53 heterozygous state. C57BL/6J mice are resistant to induced mammary tumors and develop less than 1% mammary tumors in both wild-type and p53 ^+/− states. To map modifiers of mammary tumorigenesis, we have established F₁ and F₂ crosses and backcrosses to BALB/cJ (N2-BALB/cJ) and C57BL/6J (N2-C57BL/6J) strains. All cohorts developed mammary carcinomas in p53 ^+/− females, suggesting that multiple loci dominantly and recessively contributed to mammary tumorigenesis. We mapped two modifiers of mammary tumorigenesis in the BALB/cJ strain. Mtsm1 (mammary tumor susceptibility modifier), a dominant-acting modifier, is located on chromosome 7. Mtsm1 is suggestive for linkage to mammary tumorigenesis (p = 0.001). We have analyzed the Mtsm1 region to locate candidate genes by comparing it to a rat modifier region, Mcs3, which shares syntenic conservation with Mtsm1. Expression data and SNPs were also taken into account. Five potential candidate genes within Mtsm1 are Aldh1a3, Chd2, Nipa2, Pcsk6, and Tubgcp5. The second modifier mapped is Mtsm2, a recessive-acting modifier. Mtsm2 is located on chromosome X and is significantly linked to mammary tumorigenesis (p = 1.03 × 10⁻⁷). Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.