Effects of oral administration of Pfaffia paniculata (Brazilian ginseng) on incidence of spontaneous leukemia in AKR/J mice

Pfaffia paniculata (Brazilian ginseng) administered subcutaneously and intraperitoneally inhibits growth of allogeneic cancer cells in mice. The goal of this study was to determine whether oral administration of P. paniculata inhibits development of spontaneous leukemia. Four-week-old female AKR/J mice were given oral doses of powdered roots from P. paniculata three times weekly for 8 weeks; controls received phosphate-buffered saline. Enlargement of thymic lymphoma in the mice treated with P. paniculata was significantly suppressed, as compared with controls (128 +/- 67.3 mg versus 219.9 +/- 84.2 mg, respectively; P < .01); proliferation of endogenous recombinant murine leukemia viruses (MuLV) in the thymus was markedly inhibited after the first oral treatment as compared with untreated controls (final age, 28 weeks; P < .05). In normal 3-week-old female AKR/J mice, mortality from thymic lymphoma was delayed markedly after injection into the thymus of cell-free extract of thymus from the experimental female 28-week-old AKR/J mice that received the oral P. paniculata preparation. These results suggest that the agent's suppressive effects on spontaneously occurring leukemia caused by endogenous recombinant MuLV in female AKR/J mice may depend on enhancement of nonspecific immune or cellular immune systems (or both) by the P. paniculata preparation.     

Natural antibodies directed against murine lymphosarcoma cells.

Natural antibodies reacting in a test of complement-dependent cytotoxicity with untreated murine lymphosarcoma cells of thymic origin were found in murine sera. Normal thymus cells were unaffected and unable to absorb the serum activity. The natural antibodies were IgM-like and stable at 56 degrees C. They were not uniformly distributed in the studied strains, and high (C3H/He and C3Hf), intermediate (AKR and CBA/J), and low level strains (BALB/c, DBA/2, C57BL, and C57BL/6J) were found. Hybrids between a high (C3Hf) and a low level strain (C57BL) had the same response as the parental C3Hf mice. An inverse relationship was demonstrated between cytotoxicity of, and susceptibility to, serum of lymphoma cells in a given strain, which suggested that an immunologic modulation was at work. Embryonic cells absorbed the cytotoxic activity of the normal serum.     

Accelerating effect of nude gene heterozygosity on spontaneous AKR thymic lymphomagenesis

The effect of nude gene heterozygosity on spontaneous AKR thymic lymphomagenesis was studied by comparing female littermates of AKR/Ms-nu/+ and +/+. As previously reported in nude gene heterozygotes with genetic background other than AKR, AKR/Ms-nu/+ mice had a significantly smaller thymus than the +/+ littermates. Overall incidences of thymic lymphomas were comparable in the two genotypes, but the mean latent period for lymphoma development was significantly shorter in the nu/+ mice (266.3 +/- 11.6 days) than in the +/+ mice (319.3 +/- 7.9 days). Both genotypes of mice expressed a high level of XC+-ecotropic murine leukemia virus. Expression of xenotropic virus was more variable, but there was no consistent difference in onset of virus expression or in virus titer that could explain accelerated lymphomagenesis in the nude gene heterozygotes.     

Microfluorometry of nuclear acridine orange metachromasia in lymphocytes of thymus, spleen, and blood of AKR and random-bred mice.

Lymphocyte chromatin lability to acid hyrolysis was studied using acridine orange fluorescence metachromasia in a high-lymphocytic-leukemia-susceptibility strain (AKR) and random-bred mice (ICR). Comparisons were made of blood, thymus, and spleen lymphocytes between random-bred, "normal" AKR, and leukemic AKR animals. The leukemic mice were in the stages of the disease characterized by enlarged thymus and spleen but preceding massive elevation of blood lymphocytes. The ranges of the mean chromatin acid lability overlapped and were nearly identical in peripheral blood lymphocytes. However, thymic and splenic lymphocytes showed a marked rise in mean chromatic acid lability in the leukemic animals. The ranges of the mean values of this parameter were also found to be far greater in the lymphopoietic organs of normal AKR than in the random-bred mice. The data indicate that anatomically normal AKR animals of an age in which they are highly susceptible to spontaneous lymphocytic leukemia may contain a greater number of lymphoblasts in both the spleen and the thymus than do comparable random-bred mice. The implications of these findings are discussed in relation to strain differences and the concept of thymic origin of lymphocytic leukemia in mice.     

Proviral structure and differentiation antigen phenotype of spontaneous and chemically induced AKR lymphomas

AKR mice develop spontaneous thymomas after 6 months of age due to a novel class of murine leukemia viruses that are generated by a series of genetic recombinations between endogenous proviral loci. AKR mice also are more susceptible to N-methyl-N-nitrosourea (MNU)-induced thymomas than are low-leukemia-incidence mouse strains. To determine whether virally and chemically induced lymphomagenesis proceeds by similar pathways in AKR mice, spontaneous and MNU-induced thymic lymphomas were analyzed for a DNA restriction linkage generated during spontaneous tumor development by recombination between envelope genes of endogenous murine leukemia proviral loci. DNA from spontaneous thymic lymphomas invariably contained a restriction fragment characteristic of recombinant murine leukemia virus etiology, while four of five MNU-induced thymic lymphomas did not show this restriction linkage. In addition, analysis of lymphocyte differentiation antigen profiles indicated that MNU-induced lymphomas represent a more immature stage of T-cell differentiation than the majority of spontaneous lymphomas. These data suggest that there are fundamental differences in the mechanisms of induction of virally and chemically induced thymic lymphomas in AKR mice.     

DNA and RNA analysis by flow cytometry of the leukemia in AKR mice

The development of the leukemia-lymphoma complex was studied in AKR/J (H-2k) mice using flow cytometry and staining with acridine orange. Investigation of cytokinetics and of cellular RNA content showed that during the neonatal period all mice had a significant increase of S phase cells in the thymus, bone marrow, lymph nodes and spleen reflecting extramedullary hematopoiesis. Concomitantly, G0/G1 cells were significantly reduced in the thymus, lymph nodes and spleen when compared to 6-week old mice of the same strain. No changes in the cell cycle or in RNA content were observed until 10 months of age in congeneic AKR (H-2b) mice, which do not develop leukemia during the first year of life. In leukemia-prone AKR/J (H-2k) mice, however, it was shown that the first appearance of a leukemic process may be recognized in the thymus by a significant increase of cells in G1 phase of the cell cycle which have a high RNA content. These changes were first seen at 5 months of age before the increased expression of MuLV antigen signals preleukemic alterations at 6 months of age and long before morphological changes appear (8-10 months). Furthermore, at 6 months these mice showed a significant elevation of cells in S phase which always appeared initially in the thymus. By 10 months of age, when the mice were overtly leukemic, these changes had progressed in all lymphoid organs and in the peripheral blood. At the same time a unique population of cells was observed that was characterized by cells in S and G2M with very low RNA content. The method used is applicable to further analysis of the precise locus of development of leukemia in the thymus of AKR/J (H-2k) mice, analysis of the nature of the earliest malignant cells, and investigation of the influence of viruses in the pathogenesis of AKR leukemia.     

Genetics of N-methyl-N-nitrosourea induction of thymic lymphomas in AKR/J mice: assignment of a susceptibility gene to mouse chromosome 7

Treatment of young AKR/J mice with N-methyl-N-nitrosourea (MNU) results in the induction of a high incidence of thymic lymphomas occurring between 4 and 6 months of age. The tumor incidence is higher and the latency period is shorter than that observed in other MNU-treated mouse strains. Analysis of tumor incidence in crosses of AKR/J with C57L/J mice indicates that several genes influence the incidence and latency of MNU-induced thymic lymphomas. One of these genes appears to be tightly linked to the albino locus of chromosome 7.     

Development of virus-accelerated thymic lymphoma in AKR mice

The progression of prelymphoma cells (PLC) in the bone marrow to lymphoma cells (LC) in the thymus in the presence of the lymphomagenic retrovirus SL3-3c was studied in a model system of virus-accelerated thymic lymphoma in AKR/J mice. On the average, a single LC appears in the thymus 30 days after the neonatal ip inoculation of SL3-3c virus; 50 days later, thymic lymphoma is clinically detectable. PLC in the bone marrow and oncogenic virus in the thymus are continuously present during this period before lymphoma develops. Biologically active oncogenic virus in the thymus increases as the animal nears the time of lymphoma development. Intrathymic inoculation, but not ip inoculation, of SL3-3c virus results in accelerated thymic lymphoma in 4- to 6-week-old AKR mice. PLC defined as a population of bone marrow-derived thymocyte progenitor cells susceptible to malignant transformation by oncogenic retrovirus after homing to thymus were further studied and characterized. PLC, like normal bone marrow thymocyte progenitors, were found to be radiosensitive and glucocorticoid resistant. Thymocytes of 21- to 28-day-old AKR mice, neonatally inoculated with SL3-3c virus, were studied for PLC. They could not be detected. It is concluded that lymphoma development is the final outcome of a series of events in which bone marrow-derived thymocyte progenitors are transformed after entering the thymus by virus in the thymic environment.     

Acceleration of leukemogenesis in AKR mice by grafts, cell suspensions, and cell-free centrifugates of thymuses from preleukemic AKR donors

Grafting of 5-month-old thymuses of AKR mice into young adult AKR and (AKR x SL)F₁ recipients shortened markedly the latent period for leukemia development, but thymus grafts from 3- and 1-month-old AKR mice had no accelerating effect. Transplantation assay of early lymphomas appearing in (AKR x SL)F₁ mice revealed that almost all the neoplasms were derived from F₁ host cells. Intraperitoneal injections of thymus cell suspensions made from 5-month-old AKR mice also caused acceleration of leukemogenesis. Cell-free centrifugates prepared from 5-month-old AKR mouse thymuses had a striking accelerating activity on early development of leukemia when injected intraperitoneally into isologous newborn mice. The centrifugates from 3-month-old thymuses had an intermediate activity but the centrifugates from 1-month-old thymuses had no activity. The centrifugates made similarly from AKR mouse spleens at various time points during the preleukemic stage did not show accelerating activity. Intrathymic injection of the centrifugates from 5-month-old thymuses was more effective in accelerating leukemia development than intraperitoneal injection of the same material. These results indicate that thymuses of 5-month-old AKR mice may contain a large quantity of leukemia viruses and, further, that the latent period may represent the time necessary for the development of an effective concentration of leukemia virus.     

Aging and cancerogenesis. IV. Interrelationships among age, immune response, and tumor incidence in several strains of mice

Immune reactivity of mice with various incidences of spontaneous tumors was measured at different points in their lifespan. Cytotoxic activity of immune sera from 3- and 12-month-old mice was high or moderately high in AKR/J, SJL/J, and A/HeJ mice in which high incidences of spontaneous tumors occur by the 12th month, and in SWR/J and C3HeB/-FeJ mice in which high incidences of spontaneous tumors reportedly occur after 18 months of age. A decrease in primary antibody response accompanied old age in 4 of the 6 strains tested (including randombred Swiss ICR/Ha), but not in AKR/J or A/HeJ mice. These facts, together with previous tests of cellular response in these mice, imply that under these experimental conditions little correspondence exists between humoral and cellular response. Further, host immunity may be only secondarily implicated in neoplastic formation.     

Evaluation of 5'-nucleotidase as an enzyme marker in ovarian carcinoma

A plasma membrane ectoenzyme in mammalian cells, 5'-nucleotidase, was evaluated as a marker for ovarian carcinoma. Activities of this enzyme were determined in homogenates from normal (N = 17) and malignant ovaries (N = 17), as well as in the sera from control women (N = 35), ovarian cancer patients with active disease (N = 24), and those in clinical remission (N = 9). A significant reduction of the activity of 5'-nucleotidase was observed in tumor homogenates compared with homogenates from normal ovaries. Levels of this enzyme in the sera of ovarian cancer patients were higher than in control women, suggesting the possibility of shedding of this enzyme from the tumor cell surface to the systemic circulation of the host. The diagnostic value of serum 5'-necleotidase levels was compared with another enzyme marker for ovarian carcinoma, viz. serum glycoprotein:galactosyltransferase. The upper limit of normal was set at 2 SD higher than the normal mean. Elevation of serum 5'-nucleotidase was observed in 12/24 (50%) patients with active disease, and 1/9 (11%) patients with clinical remission. In contrast, serum glycoprotein:galactosyltransferase was elevated in all the serum samples from patients with active disease and in none of those with clinical remission. There was some correlation between the serum levels of 5'-nucleotidase and those of glycoprotein:galactosyltransferase (0.01 less than P less than 0.05). Elevation of 5'-nucleotidase in the serum of these patients was not due to liver metastasis. Serum 5'-nucleotidase levels seem to correlate with disease status in some ovarian carcinoma patients, but in general it is inferior to serum glycoprotein:galactosyltransferase as a tumor marker.     

Role of the AKR gene locus AKv-1 in susceptibility to chemical induction of thymic lymphomas

Various strains of mice demonstrate widely differing susceptibility to chemical induction of thymic lymphomas, in both timing and incidence. In AKR mice tumors appear very early and at high incidence after a single dose of N-methyl-N-nitrosourea, while in other strains they appear later and at lower incidences. In an attempt to determine the potential role of AKR ecotropic murine leukemia virus loci in this process, congenic mice of NFS/N background, into which the highly productive ecotropic murine leukemia virus loci AKv-1 or AKv-2 has been transferred, were challenged with N-methyl-N-nitrosourea. Although they had a lower incidence of thymic lymphomas than did the parental donor AKR, the NS.AKv-1 mice had a tumor incidence twice that of NFS/N or NS.AKv-2. However, no difference in timing was noted, and these three strains demonstrated tumor appearance much later than that of AKR/N. It is suggested that the presence of the AKv-1 loci, or a gene of the closely associated genomic region, increases the number of target cells that are susceptible to N-methyl-N-nitrosourea.     

Activation of complement by spontaneous leukemic cells of AKR mice.

Complement activation via the alternative pathway by tumor cells was tested by rosette formation of human erythrocytes (HuE). Test cells were treated with C4-deficient guinea-pig serum (C4DS) in the presence of Mg⁺⁺ and absence of Ca⁺⁺, following which 10–30% of lymphoma cells and 10–17% of thymoma cells of spontaneous leukemia in AKR mice formed rosettes. The incidence of rosette formation did not change significantly after transplantation of these cells to other non-leukemic AKR mice or after in vitro cultivation. Following treatment with fresh C4DS, however, normal AKR mouse thymocytes as well as lymph-node cells contained only 3–4% HuE-positive cells. These findings suggest that the ability of non-specific activation of complement (NAC) may be increased when normal AKR mouse cells become transformed into leukemic cells and/or that the leukemic cells arise from thymus cells which possess NAC ability. This interpretation was further confirmed by the finding that a higher proportion of leukemic cells than of normal thymocytes were lysed by mouse complement in Mg⁺⁺-EGTA. The fact that NAC-positive AKR thymoma cells were lysed effectively by homologous C3H/He plasma agrees with earlier findings that in leukemic AKR mice supplemented with C5 a temporary regression of leukemia occurs.     

Uridine 5'-diphosphate-galactose:glycoprotein galactosyltransferase activity in the ovarian cancer patient.

Uridine 5'-diphosphate-galactose:glycoprotein galactosyltransferase activity was demonstrated in homogenates of normal ovary and ovarian epithelial adenocarcinomas. The specific activity of the enzyme in ovarian tumors was 3 to 5 times higher than in normal ovaries when the enzyme was assayed under identical conditions. The glycoprotein fetuin, from which terminal sialic acid and penultimate galactose were removed (fetuin minus N-acetylneuraminis acid and galactose), acted as an excellent exogenous acceptor. Galactosyltransferase from normal ovary and ovarian tumor cells had similar properties. Both required Mn2+ and Triton X-100 and had broad pH optima between 5.5 and 7. Galactosyltransferase activity was also measured in serum samples from ovarian cancer patients and normal healthy individuals in the presence of fetuin minus N-acetylneuraminic acid and galactose as exogenous acceptor. The enzyme levels were significantly elevated in the sera of ovarian cancer patients as compared to normal controls. The differences in the levels of this enzyme in the tissues and sera of normal individuals and ovarian cancer patients were not due to differential levels of the degrading enzymes such as uridine 5'-diphosphate-galactose pyrophosphatase or beta-D-galactosidase. Serial determinations were carried out on the sera of 5 ovarian cancer patients over a long period of time. The serum level of galactosyltransferase activity appeared to correlate with tumor volume as well as with the clinical status of the patient, which suggests possible leakage of the tumor enzyme into the host sera. Serial determination of this enzyme level in ovarian cancer patients seems promising in measuring tumor progression or success of therapeutic approaches.     

Lymphoma development from transplanted murine leukemia virus infected organ cultured thymuses: inhibitory effect of in vitro interferon treatment

Thymuses of 14-day-old AKR mouse embryos were infected with Gross murine leukemia virus (MuLV) and then maintained in organ culture for 3 weeks. When they were transplanted to 3-week-old (AKR X C3H)F1 mice, approximately 50% of these developed T lymphomas within 3-4 months. Most (22/23) tumors were of host, F1-hybrid, origin while only one was of donor AKR type. No clear evidence for in vitro MuLV-induced lymphoma cells was therefore obtained. Exposure of MuLV-infected embryonic thymuses to interferon during the organ culture period significantly reduced the incidence of lymphomas in mice receiving such thymus transpalnts. Interferon also prevented the appearance of detectable numbers of MuLV antigen-containing lymphocytes in infected organ-cultured thymuses. In contrast, despite the use of very high interferon concentrations, no effects were seen on the number of viable thymic lymphocytes, their proliferation or responsiveness to the polyclonal T-cell mitogens concanavalin A (Con A) and leukoagglutinin (LA). Thus interferon, presumably through an antiviral effect, can limit the MuLV infection in the thymus and its consequence, i.e. development of a lymphoma.     

Enhanced AKR leukemogenesis by the dual tropic viruses. I. The time and site of origin of potential leukemic cells

The occurrence of potential leukemia cells (PLC) among bone marrow, spleen, and thymus of AKR mice during the preleukemic period was tested by an in vivo transplantation bioassay. The presence of PLC in 30- and 75-day-old AKR mice was demonstrated mostly among bone marrow cells, less in spleen, and was lacking in thymus. Occurrence of PLC in young AKR mice was shown to be thymus independent. However, progression of PLC from young donors (14-80 days old) into overt leukemia following transplantation into F1 recipients was shown to be dependent on specific host conditions including an intact thymus and an Fv-1nn allele. In contrast, PLC from 7-9-month-old AKR mice or frank leukemic cells when transplanted grew in any intact or thymectomized histocompatible host, thereby indicating their autonomous growth state. Infection of 2-week-old AKR mice with the dual-tropic virus DTV-70 induced characteristic changes in the thymus and accelerated leukemia development. DTV-70 inoculation into 14-day-old AKR mice did not change the spontaneous PLC distribution pattern in the tested host organs within 30 days postinfection, nor did it change PLC-specific host requirements for further progression into leukemic cells; however, it enhanced PLC transition to autonomous leukemic cells. The preferential cell tropism of DTV-70 for target cells (prothymocytes) among bone marrow and young spleen cells rather than for thymocytes was also demonstrated in an in vitro-in vivo test. The dual tropic virus may act as a promoter on preexisting PLC (present mostly among bone marrow cells) by enhancing their ability to progress into autonomous leukemic cells.     

Comparison of sister chromatid exchanges in spleen and thymus lymphocytes from AKR, C57BL/6N x DBA/2J F1, and CBA mice following in vivo exposure to N-methyl-N-nitrosourea

The induction of sister chromatid exchange (SCE) by N-methyl-N-nitrosourea (MNU) was studied in spleen and thymus lymphocytes from AKR mice, which are highly susceptible to MNU-produced thymomas, CBA mice, which are much less sensitive to induction of thymomas by MNU, and C57BL/6N x DBA/2J F1 mice. MNU produced dose-related increases in SCE in concanavalin A (Con A)- and lipopolysaccharide-stimulated spleen lymphocytes and Con A-stimulated thymus lymphocytes from each mouse strain at 1 and 24 h posttreatment. MNU-induced SCE were generally higher in Con A-stimulated spleen lymphocytes compared to lipopolysaccharide-stimulated spleen lymphocytes and Con A-stimulated thymus lymphocytes from each mouse strain. On the whole, MNU-produced SCE were lower in AKR and CBA spleens than in C57BL/6N x DBA/2J F1 spleens. In addition, MNU-induced SCE levels in thymus lymphocytes from all three strains of mice were, for the most part, similar. These results indicate that if differences in MNU-induced genotoxicity in AKR, CBA, and C57BL/6N x DBA/2J F1 thymus lymphocytes exist, these differences cannot be ascertained by use of the in vivo/in vitro SCE assay.     

Elevation of 4 beta-galactosyltransferase activity for paragloboside synthesis in sera of patients with cancer.

Galactosyltransferase activities in sera of cancer patients were determined by assaying the formation of paragloboside from UDP-galactose and lactotriaosylceramide immobilized on microtiter plates by means of the enzyme-linked immunosorbent assay using a monoclonal antibody, H-11, directed to paragloboside. Enzyme properties were as follows. Optimum pH was 6.8 in cacodylate buffer, and Km values were 2 microM for lactotriaosylceramide and 29 microM for UDP-galactose. The enzyme activity was inhibited by the addition of alpha-lactalbumin. Glucose (20 mM) inhibited the enzyme activity in the presence of alpha-lactalbumin (0.1 mg/ml) but not in its absence. These enzyme properties are similar to those of bovine milk galactosyltransferase, indicating that the enzyme in the sera might be lactose synthetase. The enzyme activities in sera from patients with cancer, patients with benign disease, or a reference sample group were assayed. The activity was below the limit of detection (5.5 pmol/25 microliters serum/2 h) in the reference sample group. Remarkable elevations of the enzyme activity were observed with high incidence in patients with cancer, especially those with blood cancer (100%). A high incidence was observed in the progressive stage, and the enzyme activity was detected at stage 1 in lung, esophagus, stomach, colorectal, and testis cancer. The enzyme activity in sera from patients with benign disease was elevated in 22% of the patients. After effective therapies, the enzyme activity decreased to below the limit of detection. Release of the galactosyltransferase into culture medium of cancer cells could be demonstrated. These observations suggest that the galactosyltransferase is released from cancer tissue into the circulation. The present method for the assay of galactosyltransferase may be useful for the detection of patients with cancer and for monitoring neoplastic recurrence after therapy.