Studies on Murine Sarcoma Virus: antigenic characterization of Murine Sarcoma Virus induced tumor cells

The antigenicity of Murine Sarcoma Virus (MSV) transformed mouse, rat and hamster cells has been studied by several techniques designed to detect surface antigens. Antigens were readily demonstrated on neoplastic mouse and rat cells. There was complete cross-reactivity between cells induced by the Harvey (MSV-H) or Moloney (MSV-M) strains of MSV and by Moloney Leukemia Virus (MLV). No evidence of a distinct or separate ?MSV-non-MLV”? antigen could be obtained by cross absorptions of MSV/MLV antisera and cells, or by tests designed to break MLV tolerance. Such results favor the view that MSV and MLV are closely related entities. With the 8303 line of MSV-M transformed hamster cells serological tests revealed little if any reactivity with strongly positive mouse anti-MSV/MLV antisera. In one test, however, immunization of mice with these cells gave some degree of protection against syngeneic MSV sarcoma.     

Effect of Recombinant Human Interleukin 2 on the Growth of a BALB/c Sarcoma Induced by Moloney Murine Sarcoma Virus

The effect of in vivo administration of recombinant interleukin 2 (rIL2) on the growth of a primary female BALB/c sarcoma induced by Moloney marine sarcoma virus (M-MSV) was studied. Although low-dose administration of (6,000 JU/mouse × 14 days) rIL2 had no effect on the growth of the tumors, high-dose (15,000–80,000 JU/mouse × 14 days) intraperitoneal inoculation of rIL2 induced tumor regression, dose-dependently. Tumors in mice which received 80,000 JU/mouse/day of rIL2 regressed completely 2 weeks after the initiation of treatment. The survival rates of the treated groups were significantly higher than those of the control group. A time course experiment disclosed that the effect of rIL2 was restricted only to the group in which rIL2 treatment started 8 days after the inoculation of M-MSV. The cytotoxic activity of regional lymph node lymphocytes from rIL2-treated mice was demonstrated against primary culture of M-MSV-induced sarcoma but not against syngeneic tumor induced by methylcholanthrene (Meth A). The effect of rIL2 was partially blocked by the administration of anti-IL2 receptor antibody. Immunohistochemical examination revealed that infiltration of Thy1.2⁺Lyt1⁺2^- (helper/inducer subset) lymphocytes into the tumor tissue was prominent in mice which received high-dose rIL2. The results indicated that IL2 induced regression of M-MSV-induced sarcoma mainly through activation of IL2-receptor-positive helper T cells in the tumor tissues and of killer cells in the draining lymph nodes.     

Heterogeneity of plasminogen activator expression in various Moloney virus-induced tumor cell lines. Lack of correlation with tumor growth and cell phenotype

The aberrant expression of a plasminogen activator (PA) by Moloney virus (MuLV)-transformed mouse lymphocytes and its relation to cell phenotype and tumor growth have been studied. Nine cultured cell lines were established from neoplastic splenic and thymic tissues obtained from B10 congeneic mice inoculated with MuLV and killed when overtly leukemic. Cell surface markers were assayed by microcytotoxicity tests, the concentration of MuLV p30 and group-specific MuLV gp 70 was determined by radioimmunoassays and the expression of PA activity was assessed in a fibrin-agar plate method. PA activity of 24 h serum-free culture supernatant, intact cells or cell lysates (2 X 10(5) cells/ml) was expressed in International Units by reference to a urokinase standard curve. Tumor extension and cell morphology were investigated by histologic and cytomorphologic analysis. In all cases the cell lines were derived from T cells. PA activity is not expressed by normal lymphocytes, but variations in PA expression were observed in the transformed cells. Five out of nine transformed cell lines showed PA activity with a range of 1.3 to 9.9 IU/ml. No PA activity could be detected in the other cell lines. No correlation was found between PA expression and the cell-surface-expressed phenotype, neither was there any correlation between the PA content, the cytopathological features and the degree and type of organ infiltration. This lack of correlation indicates that there is no relation between PA activity and the expression of the transformed phenotype, and that the presence of PA activity seems to be irrelevant to the tumorigenic capacities of the transformed cell lines.     

Murine sarcoma virus (Harvey): characteristics of focus formation in mouse embryo cell cultures, and virus production by hamster tumor cells

Titrations of the Harvey isolate of murine sarcoma virus (MSV) in mouse embryo cells indicated that infection of a cell with MSV alone might not be sufficient to induce focus formation. Addition of high concentrations of Moloney leukemia virus to diluted MSV preparations resulted in an increased number of foci, consistent with the hypothesis that Moloney leukemia virus can act as a helper for defective MSV (Harvey). A transplantable hamster tumor line was established by inoculation of fragments from an MSV-induced mouse tumor into newborn hamsters, followed by repeated passage in adult hamsters. Tumor tissue from the 25th hamster transplant generation of this tumor line was used to establish a cell line — “B-34” — in culture. B-34 cells elaborated a filtrable agent which produced sarcomas and other lesions characteristic of MSV when inoculated into newborn hamsters, but which did not produce any observable pathological changes when inoculated into newborn mice. However, mixed cultures of B-34 cells and normal mouse embryo cells, when superinfected with Moloney leukemia virus, released MSV infective for both mice and mouse embryo cell cultures.     

Characterization of Tu-2449, a glioma cell line derived from a spontaneous tumor in GFAP-v-src-transgenic mice: comparison with established murine glioma cell lines.

Glial fibrillary acidic protein (GFAP)-v-src transgenic mice develop spontaneous gliomas with a high incidence of malignant progression. We characterize the first glial cell line derived from v-src transgenic mice, Tu-2449 in comparison with a virally induced murine glioma, SRB-10, and a spontaneous murine glioma, P497. Doubling times were lowest, as clonogenicity in soft agar was highest for Tu-2449, and to a lesser degree, Tu-2449 cells formed spheroids and showed migratory behaviour and invaded fetal rat brain aggregates. BCL-2 and BAX expression were lower in Tu-2449 and P497 than in SRB-10 cells. Only Tu-2449 cells accumulated p53 protein in response to genotoxic stress. Tu-2449 and SRB-10 cells that showed low CD95 expression were resistant to CD95 ligand (CD95L)-induced apoptosis unless coexposed to CD95L and inhibitors of RNA or protein synthesis. A chemosensitivity profile revealed Tu-2449 to be rather chemoresistant. Tu-2449 thus displays growth characteristics and patterns of resistance to apoptosis similar to those of other mouse and human glioma cell lines and may therefore become a valuable tool to evaluate new therapies for malignant gliomas in vitro and in vivo.     

In vitro comparison of Essiac and Flor-Essence on human tumor cell lines

Essiac (ES) and Flor-Essence (FE) are two herbal teas widely taken by North American cancer patients during chemo- and radiation therapy. In vitro studies on the antiproliferative and differentiation inducing activities of these teas were performed. ES and FE showed negligible antiproliferative activity on Jurkat leukemia cells. Both herbal teas inhibited 50% (IC50) of MCF7 breast cancer cell growth at 1/10 dilution. The IC50 was about 1/40 and 1/10 dilution of FE and ES respectively for MDA-MB-468 human breast cancer cells. The IC50 for HL60 cells was at 1/10 dilution of FE and less than 1/10 dilution of ES. ES at 1/10 dilution induced expression of non-specific esterase in 16% of HL60 cells, compared to about 5% in FE treated cells and untreated controls. ES treatment of HL60 cells induced 47-67% nitroblue tetrazolium positive staining cells compared to 24.6+/-3.1% in cells treated with 1/10 dilution of FE. Flow cytometry analysis showed that both ES and FE treatment between 1/10 and 1/100 dilutions only slightly affected the cell cycle progression of MCF7, MDA-MB-468, Jurkat and HL60 cells. Our data show that both ES and FE herbal teas demonstrated antiproliferative and differentiation inducing properties in vitro only at high concentrations. Further research is needed to elucidate the in vivo activities.     

Two-way selection of a stock of Swiss albino mice for mammary tumorigenesis: establishment of two new strains (SHN and SLN).

Two mouse strains (SHN and SLN) with high and low incidences, respectively, of mammary tumors were established from the same basal stock of Swiss albino mice that were unrelated in origin to other mouse strains with mammary tumors. In the breeders, mammary tumor incidence and average age of the mice when they developed mammary tumors were 97.2 % and 6.6 months for SHN, and 5.57% and 10.1 months for SLN, respectively...     

Unknown primary tumors: establishment of cell lines, identification of chromosomal abnormalities, and implications for a second type of tumor progression

We describe the establishment of two human unknown primary tumor (UPT) cell lines and a comparison of their cytogenetic properties with two direct harvests from two tumor biopsy specimens. Histopathological analysis of all four tumor specimens revealed them to be undifferentiated adenocarcinomas of unknown origin. The chromosome numbers in these samples vary between 38 and 144. Consistent structural anomalies involving chromosome 1 were observed in all cases. These included a deletion of the short arm (4 of 4), duplication of 1q (3 of 4) and the presence of homogeneously staining region (2 of 4). Several additional chromosomal changes involving chromosomes 7, 8, and 9 were also observed, but these were less consistent and their importance is not yet clear. Abnormalities in chromosome 1 have generally been associated with advanced malignancy. The finding of consistent chromosome 1 changes in UPT supports our hypothesis that these are type 2 progressors in that benign or less malignant stages are not readily identified. In short, UPT are likely malignant soon after transformation occurs, and this is reflected in their rapid acquisition of the metastatic phenotype and abnormalities in chromosome 1. We are therefore characterizing a number of additional UPT cases to determine if chromosome 1 and other cytogenetic changes are consistently associated with this unique subgroup of tumors.     

Antitumor effect of docetaxel against human esophagus tumor cell lines and tumor xenografts in nude mice

The antitumor effect of docetaxel against cultured human esophagus tumor cell lines and tumor xenografts in nude mice was investigated. In the in vitro study, docetaxel showed concentration-dependent inhibition of the growth of 4 tumor cell lines having different degrees of differentiation (T. T, TE-5, TE-9 and TE-15) with IC(50) values ranging from 0.84 to 1.68 ng/ml when exposed for 72 h. These values represent ca. 1/2,700-1/1,400 of the mean maximum plasma concentration of 2.27 microg/ml attained in the clinical setting. In addition, the activity was found to be ca. two-fold stronger than that of paclitaxel, and much more potent than fluorouracil and cisplatin. The in vivo antitumor effect of docetaxel was also investigated against xenografts of human esophagus squamous cell carcinoma H-190 (highly differentiated) and H-204 (moderately differentiated) in nude mice. Docetaxel at its Maximum Tolerated Dose (MTD) and the lower dose (4.5, 6.7 mg/kg/dose, q 4 d x 3, iv) showed a significant growth inhibition of ca. 100% against H-190 tumor, resulting in the tumor shrinkage. Paclitaxel (6.7, 10 mg/kg/dose, q 4 d x 3, iv) showed a tumor-shrinking effect similar to that seen with docetaxel. In the H-204 xenograft model, docetaxel (4.5, 6.7, 10 mg/kg/dose) exhibited a dose-dependent effect in delaying the tumor growth, while paclitaxel failed to suppress the tumor growth even at its MTD. These results demonstrated that docetaxel has potent antitumor efficacy against human esophagus tumor cells, leading to the expectation that it will be useful as a therapeutic agent for esophagus cancer.     

Gene for ovarian granulosa cell tumor susceptibility, Gct, in SWXJ recombinant inbred strains of mice revealed by dehydroepiandrosterone

Spontaneous, malignant ovarian granulosa cell (GC) tumors occur in pubertal SWR and specific SWXJ recombinant inbred strains of mice. Treatment of these mice with dehydroepiandrosterone (DHEA), an adrenal secretory steroid with anticancer actions against spontaneous and carcinogen-induced tumors of different tissues, gave unexpected results. Diet supplemented with 0.4% DHEA (a) induced significantly more GC tumors in spontaneous tumor-susceptible strains (SWR and SWXJ-1, -4, and -9), (b) induced the first GC tumors observed in five previously tumor-free strains (SWXJ-6, -7, -8, -10, and -12), and (c) failed to induce GC tumors in SJL and in the remaining six SWXJ strains (SWXJ-2, -3, -5, -11, -13, and -14). The strain distribution pattern of DHEA-induced GC tumor susceptibility versus resistance was compared with strain distribution patterns for 35 different loci known to distinguish SWR and SJL progenitor strains. A complete match of DHEA-induced GC tumors with pancreas-2 (Pan-2) on mouse chromosome 4 was found. We have named this new locus GC tumor susceptibility (Gct), with the Gcts (susceptible) allele found in SWR and the Gctr (resistant) allele found in SJL mice. The Gct locus is closely linked to pancreas-2, Pan-2, but the order of genes is not yet confirmed. In addition, data from F1 progeny of matings between SWR and selected inbred strains provide suggestive evidence for a second gene controlling GC tumor incidence that we hypothesize involves steroid metabolism. Differences in GC tumor incidence data from reciprocal F1 progeny of matings between SWR and SJL mice reveal a strong maternal effect that may represent yet a third gene. These data support a heritable basis for GC tumorigenesis in the SWR model involving a small number of genes.     

The effects of human recombinant tumor necrosis factor on glioma-derived cell lines: cellular proliferation, cytotoxicity, morphological and radioreceptor studies

To determine whether tumor necrosis factor is of potential value for the treatment of human malignant gliomas, we studied the effects of human recombinant tumor necrosis factor (rTNF-alpha) on the morphology, incorporation of tritiated thymidine, and proliferation of 5 established cell lines derived from human malignant gliomas and 3 normal human brain cell cultures. A radioreceptor analysis for rTNF-alpha was performed on all cell lines and cultures. Two of the 5 human glioma cell lines (SF-188 and U 343 MG-A) demonstrated a marked decrease (60% or less of untreated controls) in the uptake of tritiated thymidine when treated with rTNF-alpha at a concentration of 40 U/ml; rTNF-alpha at 100 U/ml had antiproliferative and cytotoxic effects on both cell lines. The growth and proliferation of cell lines SF-126 and U 251 MG were not affected by rTNF-alpha even at high concentrations (5,000 U/ml). The growth and proliferation of SF-539 were affected to an intermediate degree. A colony-forming efficiency assay corroborated the results of the proliferation studies: SF-126 was relatively resistant (surviving fraction of 0.9 at 500 U/ml) and SF-188 was relatively sensitive (surviving fraction of 0.08 at 500 U/ml) to the cytotoxic effects of rTNF-alpha. Time-sequence electron microscopy showed that rTNF-alpha at a concentration of 500 U/ml caused ultrastructural changes in SF-188, including increased intracytoplasmic vesiculation, swelling and degeneration of mitochondria, loss of cell:cell junctional complexes, and fragmentation of the plasma membrane. Studies with 125I-rTNF-alpha showed a variable degree of binding in all cell lines and cultures. SF-188, a highly sensitive cell line, demonstrated the strongest binding of 125I-rTNF-alpha (3,400 receptors/cell with high affinity; kd = 0.27 nM), while SF-126, a highly resistant cell line, had the weakest binding (809 receptors/cell; kd = 0.25 nM). We conclude that there is a spectrum of antiproliferative and cytotoxic activity among glioma-derived tumor cell lines exposed to rTNF-alpha. An increased number of rTNF-alpha receptors appears to be a necessary but insufficient condition to explain the antiproliferative effects observed in some glioma-derived cell lines.     

Engraftment and tumorigenesis of HTLV-1 transformed T cell lines in SCID/bg and NOD/SCID mice

Human T cell leukemia/lymphoma virus type-1 (HTLV-1) is recognized as the etiological agent of adult T cell leukemia (ATL). Although HTLV-1 can immortalize human lymphocytes in culture, identification of molecular events leading to tumorigenesis after HTLV-1 infection remain elusive. SCID/bg and NOD/SCID mice have reduced natural killer (NK) cell activity and were inoculated intraperitoneally with HTLV-1 transformed cells to refine and characterize the SCID mouse as a small animal model for investigation of HTLV-1 tumorigenesis. HTLV-1 transformed cell lines originally derived by cocultivation of uninfected peripheral blood mononuclear cells (PBMC) with lethally irradiated leukemic cells from patient samples (SLB-1, MT-2 and HT-1-RV) were lymphomagenic when inoculated into NOD/SCID mice. In contrast, immortalized cell lines generated by transfection PBMC with an infectious molecular clone of HTLV-1 (ACH or ACH.p12) were not tumorigenic. The differing behaviors of HTLV-1 infected cell lines in NOD/SCID mice indicates that viral infection and immortalization of human PBMC for growth in culture is not sufficient for induction of a tumorigenic phenotype. The higher level of engraftment of HTLV-1 transformed cell lines in NOD/SCID mice suggests that this is an effective animal model to investigate molecular determinants of HTLV-1 lymphomagenesis.     

The growth and metastasis of human, HER-2/neu-overexpressing tumor cell lines in male SCID mice

HER-2/neu is overexpressed on a variety of human adenocarcinomas and overexpression has been associated with a poor prognosis. For this reason, HER-2 has become an attractive target for immunotherapy. To facilitate testing of anti-HER-2-monoclonal antibodies (MAbs) and immunotoxins (ITs), we have evaluated the in vivo growth and metastatic spread of three HER-2-overexpressing human breast cancer cell lines (BT474, MDA-MB-453 and HCC1954) and one ovarian cancer cell line (SKOV3.ip1) in pre-irradiated male SCID mice using subcutaneous (s.c.), intravenous (i.v.) and intraperitoneal (i.p.) routes of injection. All the cell lines tested grew as s.c. tumors and the growth of BT474 and MDA-MB-453 cells after s.c. injection was improved by co-inoculation with Matrigel. Metastases to the lungs were detectable by PCR or histopathology after s.c. injection of BT474 and to a much lesser extent after s.c. injection of HCC1954, MD-MB-453 and SKOV3.ip1cells. I.P. injection of HCC1954 and SKOV3.ip1 cells produced fatal ascites while i.v. injection of SKOV3.ip1, but not BT474 or MDA-MB-453 cells, resulted in infiltration of lungs and death within 9–11 weeks.     

Variation in selectivity of tumor cell cytolysis by murine macrophages, macrophage-like cell lines and NK cells

Several murine tumor-cell lines were tested by isotope release assays for their susceptibility to lysis by either activated peritoneal macrophages (apMPh), macrophage-like (MPh-like) cell lines, or natural killer (NK) cells. The qualitative selectivity of tumor-cell lysis by these different effector cells was quite disparate. The rank order of target cell susceptibility to lysis by apMPh in 24 h assay was L5178Y greater than P815 approximately equal to RL male greater than YAC-1 approximately equal to MBL-2. This was seen regardless of whether peritoneal MPh (pMPh) were activated by LPS or poly I:C. Two MPh-like cell lines, PU-5R and FC-1, had a pattern of cytotoxic activity against these target cells that closely paralleled that associated with apMPh, although levels of reactivity differed quantitatively among the effector cells. In contrast, the MPh-like cell line RAW-264 expressed a qualitatively different pattern of target-cell selectivity, preferentially lysing MBL-2, which was relatively refractory to lysis by other MPh-like cell lines or apMPh in the 24 h cytolytic assay. When spontaneous or interferon (IFN)-augmented NK activity was measured against the same panel of target cells, the pattern of selectivity was qualitatively different from that observed for apMPh. The consistent rank order of susceptibility to lysis by NK cells was YAC-1 greater than RL male 1 greater than P815 approximately equal to L5178Y approximately equal to MBL-2. The characteristic patterns of target-cell selectivity for apMPh or NK cells were the same for all of the strains of mice tested. From the different selectivity patterns of apMPh and NK cells, it is concluded that lysis of target cells is not based solely on inherent sensitivity to cytolysis. Instead, selectivity of lysis is probably due to variations in expression of target-cell structures recognized by each type of effector cell, and/or in susceptibility to the lytic mechanism(s) of the various effector populations.     

ADVANCES IN THE STUDY ON LEUKEMIA STRAINS AND LEUKEMIA CELL LINES IN CHINA

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周期蛋白6在DNA复制、细胞周期检测点和肿瘤发生发展中的作用

周期蛋白6(CDC6)是组成前复制复合物(Pre-RC)的主要蛋白之一,控制细胞从G1期进入S期,同时也参与激活和维持有丝分裂S-M期检测点机制.最近的研究发现其也具有原癌基因的特性,并在人多种肿瘤细胞中存在高表达,对肿瘤发生发展起重要作用.CDC6致癌机制可能与INK4/ARF连结物信号途径和(或)某些替代机制有关.