Clonal transformation of human leukocytes by Epstein-Barr virus in soft agar.

The B95-8 strain of Epstein-Barr virus (EBV) induced colony formation of human umbilical cord-blood leukocytes in soft agar medium. One-hit response relationship between the number of colonies and the virus dose was observed with high dilutions of the virus preparation. However, there was a presumed cell-killing effect with low dilutions of virus. The colonies were similarly induced, but with a lower efficiency, in adult peripheral blood leukocyte cultures infected with the virus. The colony-forming activity of EBV was neutralized by anti-EBV-positive but not by negative human sera. The cells in colonies were capable of growing continuously and carried EBV-associated nuclear antigen. Thus, it was evident that the colony formation was caused by clonal transformation by EBV.     

Colonies of EBNA-positive cells in soft agar from peripheral leukocytes of infectious mononucleosis patients.

Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA)-positive lymphoblastoid cells grew as colonies in soft agar after seeding of leukocytes from the peripheral blood of four patients with infectious mononucleosis serologically determined to be caused by EBV. In individual cases more colonies were obtained from blood specimens during the acute phase of the disease than during the convalescent phase. Incorporation of human umbilical cord serum, which contained neutralizing antibody to EBV, into the agar medium did not reduce the number of colonies developing. Our observations indicate that colony-forming cells were originally present in the blood samples, and that they were not infected and subsequently transformed in vitro. Cells from less than 20% of the EBNA-positive colonies grew to form lymphoblastoid cell lines, which were EBNA-positive and had B lymphocyte surface markers. However, the majority (over 80%) of the EBNA-positive colonies failed to form immortalized cell lines. No colonies were obtained from 91 blood samples from healthy young adults and from five patients with an IM-like disease unrelated to EBV infections. The present results strongly suggest that already transformed cells or cells very easily transformed by EBV are present in the blood of IM patients.     

Transformation by inorganic arsenic compounds of normal Syrian hamster embryo cells into a neoplastic state in which they become anchorage-independent and cause tumors in newborn hamsters

Arsenic is a known human carcinogen, but little evidence exists for its carcinogenicity in animals. In order to investigate the ability of inorganic arsenics to transform normal cells into a neoplastic state, mass cultures of normal, diploid Syrian hamster embryo (SHE) cells exposed to various concentrations of sodium arsenite or sodium arsenate for 48 hr were continually passaged and tested for neoplastic transformation, as determined by anchorage-independent growth in semisolid agar and tumorigenicity in newborn hamsters. Twenty-one of 22 (96%) untreated, control cultures senesced by 20 passages. While 1 culture escaped senescence, it did not acquire the ability to either grow in semisolid agar or form tumors in animals. Ten of 14 (71%) cultures exposed to sodium arsenite or sodium arsenate escaped senescence. Nine of the 10 (90%) arsenic-treated immortal cultures acquired the anchorage-independent phenotype. Five of 5 anchorage-independent cultures examined were tumorigenic. Two of 3 morphologically transformed colonies induced by sodium arsenate also acquired the ability to grow in semisolid agar when isolated. Amplification of the c-myc or c-Ha-ras oncogene was detected in 3 of 5 and 4 of 5 tumorigenic cell lines, respectively. Both c-myc and c-Ha-ras were amplified even in a preneoplastic, anchorage-dependent cell line, but neither was amplified in 6 of 9 anchorage-independent cell lines. Overexpression of c-myc and c-Ha-ras mRNA was observed in most of the neoplastically transformed cell lines but not in the preneoplastic cell line. Experiments using the methylation-sensitive restriction endonuclease isoschizomers HpaII and MspI revealed hypomethylation of c-myc and c-Ha-ras in the 5'-CCGG sequence of arsenic-exposed cell lines but not in the parental SHE cells or a spontaneously transformed cell line. Thus, inorganic arsenics induce neoplastic transformation of normal, diploid mammalian cells. Overexpression of oncogenes by DNA hypomethylation may participate in the arsenic-induced neoplastic transformation of mammalian cells.     

Multistage progression of Abelson virus-infected murine pre-B-cells to the tumorigenic state

The tumorigenic potential of pre-B-cells at different stages of Abelson murine leukemia virus-induced transformation was determined. Cell lines with low growth potential in liquid culture were found (a) to have a dose-dependent growth requirement for conditioned medium obtained from bone marrow cultures, (b) to have low colony-forming ability in semisolid medium in the absence of conditioned medium, and (c) to be nontumorigenic when inoculated into syngeneic mice. Culture of the factor-dependent cells in vitro leads to the emergence of factor-independent variants, which eventually dominate the population by overgrowth. Cell lines that acquired a factor-independent phenotype were able to form colonies in semisolid medium and form tumors when inoculated into syngeneic mice. These results suggest that Abelson murine leukemia virus is sufficient to initiate transformation in the infected cell but that an additional genetic alteration is needed to confer tumorigenicity.     

Regulation of granulocyte and macrophage colony-stimulating factor production by phytohaemagglutinin-treated blood mononuclear cells

The colony-stimulating activity (CSA) of medium conditioned by phytohaemagglutinin (PHA)-stimulated blood mononuclear cells was tested using granulocyte-macrophage colony-forming cells (GM-CFC) from normal bone marrow. Low concentrations of the conditioned medium stimulated granulocytic colony-forming cells (CFC) which formed colonies by the seventh day of incubation; higher concentrations stimulated the formation of macrophage colonies which were not seen until the end of the second week in culture.The colony-stimulating activities could not be demonstrated in adherent cell-depleted bone marrow cultures. This dependence of activity on adherent cells was confirmed by incubating different concentrations of conditioned medium with isolated adherent cells and then testing for colony-stimulating activity in cultures of non-adherent bone marrow cells. The activities of conditioned media following exposure to adherent cells corresponded to the results seen when the conditioned medium from PHA-stimulated mononuclear cell cultures was used to stimulate agar cultures of unseparated marrow. The results suggest that PHA-responsive mononuclear cells (probably T lymphocytes) may modulate the regulation of colony-stimulating factor (CSF) production by adherent colony-stimulating cells (CSC).     

Epstein-Barr virus-induced transformation of human leukocytes after cell fractionation.

The efficiency of transformation of human lymphocytes after infection with Epstein-Barr virus (EBV) was determined in fractionated and non-fractionated preparations derived from 16 human cord blood samples and two blood samples from adult donors. The transformation efficiency of macrophage-depleted leukocytes was consistently lower as compared to non-fractionated leukocytes. Additional depletion of B-cells resulted in a further decrease. Reduction of T-cells, however, did not influence significantly the transformation rate. In non-fractionated leukocyte cultures, as well as in macrophage-depleted and B-cell enriched cultures, colonies of transformed cells were regularly observed within the first week of cultivation. All cell lines established after EBV-infection revealed membrane-bound immunoglobulin. Reconstruction of macrophages-depleted, B-cell enriched or B-cell depleted cultures with autologous macrophages resulted in an increase of the transfromation efficiency up to the values of non-fractionated leukocyte preparations. Addition of heterologous human embryonic lung fibroblasts resulted in a similar increase. The results support the interpretation that EBV transforms only those cells of the hematopoetic system which are derived from the bone-marrow entity. The transformation efficiency is considerably increased by co-cultivation of lymphocytes with macrophages and heterologous human fibroblasts which seem to excert a feeder-layer effect by enhancing survival of lymphocytes in vitro.     

Generation in vitro of EBV-induced specific cytotoxic T cells in autologous serum avoids complications due to self-preferred foetal calf serum-specific T-cell cytotoxicity

A previous report has established that in cultures of human mononuclear leukocytes, foetal calf serum (FCS) is capable of generating high levels of T cells preferentially cytotoxic for the autologous lymphoblastoid cell line (LCL). The present study compared the capacity of Epstein-Barr virus (EBV) to generate cytotoxic T cells in cultures of mononuclear cells grown in FCS in this system. Five EBV-seropositive and three seronegative donors were used and cultures were harvested at 14 days. With cultures from seropositive donors, whether grown in FCS or in autologous serum, EBV infection generated T cells cytotoxic for the autologous LCL; the response in uninfected control cultures was markedly lower. With seronegative donor cultures grown in FCS, there was virtually no difference in the capacity of T cells generated in infected or uninfected cultures to lyse the autologous LCL. Moreover, cells from seronegative donors cultured in human serum gave no detectable lysis of autologous LCL in either infected or uninfected cultures, clearly showing the absence of a response to EBV. This evidence shows that it is possible to distinguish the generation of specific cytotoxic T cells by FCS from generation by EBV, and with certain donors the apparently EBV-induced response may actually include a significant component induced by FCS in the medium. The cytotoxicity patterns of EBV-induced and FCS-induced T cells for autologous and allogeneic LCL targets showed a degree of parallelism, stressing the need for caution in interpretation of data obtained from cultures using FCS.     

鼻咽癌细胞系SUNE中EBV-LMP1基因对上皮细胞增殖的影响

目的研究鼻咽癌细胞系ＳＵＮＥ中ＥＢＶ┐ＬＭＰ１基因对上皮细胞增殖的影响,探索ＬＭＰ１在鼻咽癌发生中所起的作用。方法用ＬＭＰ１基因真核表达质粒转染人胚肾上皮细胞,检测ＬＭＰ１的表达,观察细胞在软琼脂中的集落形成能力,ＭＴＴ吸收能力以及ＰＣＮＡ的表达情况。结果被ＬＭＰ１基因转染的细胞生长旺盛,能在软琼脂中形成多个集落,ＭＴＴ吸收能力增强,ＰＣＮＡ的表达水平增高。结论ＬＭＰ１基因能明显改变上皮细胞的生物学行为,促进细胞的生长、增殖和转化,使转染的上皮细胞获得肿瘤细胞的生长特征。     

Growth in semisolid agar of prostate cancer cells obtained from bone marrow aspirates

Thirty-one bone marrow aspirations were performed on patients with prostatic carcinoma metastatic to bone. After separation over a Ficoll-Hypaque gradient viable nucleated cells were cultured in semisolid agar. Colony formation occurred in 14 of 27 (52%) nonbacterially contaminated cultures. Characterization of cells from the colonies showed them to be consistent with malignant prostate cells. After staining, these cells were periodic acid-Schiff positive, prostatic acid phosphatase positive, and prostatic specific antigen positive. Other studies demonstrated the cells to be karyotypically abnormal, ultrastructurally similar to epithelial cells, and capable of secondary colony formation. Three bone marrow aspirate specimens did not have metastatic prostatic carcinoma detected by standard methods but did demonstrate colony formation. However, colony formation was most frequently seen when a radionuclide scan was positive at the aspiration site and when tumor cells were microscopically detectable by Wright staining of a smeared aspirate. The potential utility of colony forming cultures in prostate cancer is discussed. In working with bone marrow aspirates, additional cell separation procedures may be required to calculate and maximize plating efficiencies.     

Relation between colony formation in calcium-deficient medium, colony formation in soft agar, and tumor formation by T51B rat liver cells

T51B rat liver cells and several carcinogen-treated, carcinogen + saccharin-treated, or spontaneously altered clones of T51B cells were tested for their abilities to form colonies in calcium-deficient medium and soft agar and to produce tumors in athymic nude mice. Most (10 out of 11) of the clones which were derived from colonies in calcium-deficient medium were unable to form colonies in soft agar and 8 out of 11 were non-tumorigenic. Conversely, 6 out of 9 clones derived from colonies in soft agar were unable to multiply significantly in calcium-deficient medium and 5 of these 6 clones were also non-tumorigenic. Two of these 9 soft agar-growing clones were tumorigenic, one of which also proliferated in calcium-deficient medium, and the other of which acquired the ability to proliferate in calcium-deficient medium after it became able to form tumors in athymic nude mice. Thus, T51B rat liver cells gain the ability to grow in calcium-deficient medium and soft agar independently during the process of neoplastic transformation and neither characteristic by itself reliably predicts tumorigenicity.     

The effect of long-term marrow culture on the origin of colony-forming cells in acute myeloblastic leukemia: studies of two patients heterozygous for glucose-6-phosphate dehydrogenase

Long-term marrow cultures (LTMCs) provide a selective growth advantage for cytogenetically normal cells in patients with acute and chronic myeloid leukemias. In the present study, LTMCs were established from two patients with newly diagnosed acute myeloid leukemia (AML) who were heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD). Initially only leukemic clusters grew from cells plated in semisolid medium, but after 1 or more weeks in LTMC, morphologically normal granulocyte-macrophage colonies were detected. Nonetheless, in one of the patients, more than 80% of these colonies expressed the G6PD type observed in the leukemic blast cells, indicating a probable neoplastic derivation for many of them. In the second patient, colonies cultured during the first 3 weeks of the LTMC were predominantly derived from clonal progenitors, whereas after week 4 the colonies were derived from normal stem cells. Colonies derived from clonal or normal stem cells were not morphologically distinguishable. These data support the conclusion that LTMC has a selective anti-leukemic effect on marrow cells from some patients. However, normalization of colony growth is by itself not a sufficient criterion for determination of whether committed progenitor cells from patients with AML are derived from normal or leukemic stem cells.     

Differentiation and growth modulation of chronic myelogenous leukemia cells by bryostatin

We have examined the ability of bryostatin 1 (bryo), an activator of protein kinase C, to induce differentiation of chronic myelogenous leukemia (CML) cells obtained from peripheral blood. Bryo induced a prompt and persistent macrophage-like differentiation, as evidenced by functional, morphological, and immunological criteria. Differentiated cells remained viable for at least 21 days with little change in cell number. CML cell cultures treated in semisolid medium with bryo showed diffuse infiltration with single macrophages, as well as discrete macrophage, mixed, and granulocytic colonies. Supernatants of suspension cultures of bryo-treated CML cells contained granulocyte-macrophage colony-stimulating factor (GM-CSF) by enzyme-linked immunosorbent assay. Furthermore, colony formation could be significantly inhibited by the addition of antibodies to GM-CSF. Prolonged liquid culture of CML cells in bryo reduced colony-forming unit, granulocyte-macrophage content. Bryo-induced differentiation was associated with a decrease in lactoferrin, a marker of granulocyte differentiation, and an increase in both c-fms and interleukin-1 beta RNA, both of which are expressed by monocytes/macrophages. These data demonstrate that bryostatin 1 is capable of inducing macrophage-like differentiation in maturing CML cells. Furthermore, bryostatin induces secretion of GM-CSF by such cells in suspension and semisolid medium and also promotes clonal extinction of granulocyte-macrophage progenitors. Bryostatin may be a possible therapeutic agent for CML.     

Some factors influencing the behaviour of BHK 21 Cl 13 cells in soft agar medium

As part of an attempt to reproduce Styles' cell transformationassay, the effect of serum concentration on the growth of normaland 4-nitroquinoline-1-oxide (4-NQO)-treated BHK 21 Cl 13 cellsin soft agar medium was examined. Using medium with 10% newborncalf serum, dilution of control cultures from 5 x 10⁴ to 1.56x 10³ cells/ml caused little increase in the number of countable(>0.3 mm diameter) colonies, but 4NQO caused a marked dose-relatedincrease. In contrast, using 20% of the same batch of serum,4NQO-treated groups and controls diluted to comparable viabilitycounts showed very similar increases in countable colonies.Clones >0.3 mm diameter isolated from control cultures with20% serum did not appear to be transformed when grown in agarwith 10% serum. These data indicate that factors other thanneoplastic transformation can influence the growth of BHK 21Cl 13 cells in soft agar medium.     

In vitro evaluation of cell-mediated immunity to Epstein-Barr herpesvirus by cell migration inhibition tests.

Migration of peripheral leukocytes in samples from sensitized [Epstein-Barr virus (EBV) antibody-positive] humans was greatly inhibited when challenged by antigen prepared from EBV-producing P3HR-1 cells but not by antigen prepared from EBV-nonproducing RAJI cells, EBV-negative human fibroblasts, or epithelial cells. Such inhibition was not observed when peripheral leuocytes from subjects or neonates not sensitized to EBV were challenged. Similar results were obtained in a two-stage test when the same leukocyte samples were challenged in vitro by antigen prepared from P3HR-1 cells and the cell-free supernatant was assayed for migration inhibition factor (MIF) in the guinea pig macrophage migration inhibition test; migration of guinea pig peritoneal exudate cells was greatly inhibited by the supernatant filtrates of leukocyte cultures only from subjects positive for EBV-antibody. Furthermore, this inhibitory effect was not observed if supernatant filtrates from leukocyte cultures challenged by antigens prepared from RAJI cells, fibroblasts, or epithelial cells were used. The EBV antigen transformed peripheral leukocytes and induced early antigen production in RAJI cells; however, a "killed" preparation (by UV irradiation) was sufficient for eliciting MIF production.     

Effect of tumor colony definition on ionizing radiation survival curves of melanoma-colony forming cells

Definition of survival and measurement of colony size in soft agar assays is important in establishing in vitro radiation survival curves. Conventionally, survival is assessed according to colony-forming ability. The distinction between small colonies that are abortive and those that are viable often involves a difficult and arbitrary choice for the investigator. We have examined the effect of different minimum colony sizes (greater than or equal to 25, greater than or equal to 50, greater than or equal to 75, and greater than or equal to 100 cells) on ionizing radiation survival curves for cells from established murine (CCL 53.1) and human (M1RW5) melanoma cell lines as well as from short-term human melanoma cell strains (C8146A, C8146C, C8161, C83-2C, C82-7A1, and C8442) and patient biopsy (83-4). Single cell suspensions were plated in the upper layer of the agar bilayer and cells were irradiated by single dose X rays. Giant cells did not form in colonies containing 50 or more cells. D0 values were highest (D0 values, from 390 to 100 cGy) for cells forming smaller colonies (greater than or equal to 25 cells, greater than or equal to 4-5 doublings) and lowest (D0 values, from 190 to 50 cGy) for cells forming larger colonies (greater than or equal to 100 cells, greater than or equal to 6-7 doublings). Therefore, apparent radiosensitivity was dependent on colony size selected for analysis. Precise measurement of colony size was important in establishing radiation survival curves because errors in determining the colony size will alter apparent radiosensitivity of cells. These results should help define the biological meaning of tumor colony growth in semisolid medium, and alter the interpretation of survival curves which measure sensitivity to agents using this assay.     

Suppression by protein kinase C inhibitors of tumor promoter enhancement of Epstein-Barr virus-induced growth transformation.

The tumor promoter teleocidin activates latent Epstein-Barr virus (EBV) genomes and enhances EBV-induced growth transformation of human B cells, with an activity comparable to that of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). It has been suggested that the TPA induction of EBV genomes is mediated by protein kinase C (PKC), an enzyme closely linked to signal transduction. We examined newly isolated, highly selective PKC inhibitors, UCN-01 (7-hydroxyl-staurosporine) and calphostin C, for the possible suppression of teleocidin enhancement of cord blood B lymphocyte growth transformation by EBV. 0.2 nM teleocidin B4 enhanced EBV-induced 3H-thymidine uptake 6 times, outgrowth in limiting dilution culture 5 times, and colony formation in semisolid agar 3 times. All these events were suppressed by exposure to 10-100 nM UCN-01 or to calphostin C. Our findings suggest that the tumor promoter enhancement of EBV growth transformation is probably mediated by PKC.     

Friend virus-infected long-term bone marrow cultures produce colony stimulating factor dependent and independent granulocyte-macrophage progenitor cells for over four years in vitro

C3H/HeJ mouse long-term bone marrow cultures infected at initiation with a cloned polycythemic strain of Friend spleen focus forming virus in a cloned N-tropic murine leukemia virus helper virus coat, persistently produced: colony-forming unit spleen (CFUs) for 55 weeks that formed macroscopic spleen colonies in syngeneic or allogeneic C57B10.Br/J mice; and L-cell or WEHI-3 cell conditioned medium-dependent granulocyte-macrophage colony forming unit culture (GM-CFUc); and morphologically normal granulocytes for over 245 weeks. Colony stimulating factor (CSF)-independent colony forming progenitor cells were first detectably produced in vitro at 75 weeks, and when subcultured generated karyotypically distinct permanent factor-independent tumorigenic cell lines. Nonadherent cells removed from long-term marrow cultures at 19 but not at 77 weeks reconstituted donor origin hematopoiesis in C57B10.Br/J mice as measured by B-cell lineage surface immunoglobin allotype. Nonadherent cells removed at 77 weeks produced lethal splenomegaly and marrow infiltration with culture origin cells in C57B10.Br/J mice. Despite generation of clonal malignant cell lines, L-cell DSF (CSF-1, M-CSF) responsive GM-CFUc that were simultaneously produced over 4 years in the same long-term marrow cultures, grew to 7 day colonies in semisolid medium and terminally differentiated. Thus, adherent stromal cells in Friend virus-infected long-term bone marrow cultures simultaneously support CSF-responsive and malignant CSF-independent hematopoietic progenitor cells.     

Persisting colonies in agar cultures containing serum from patients with CML in blastic transformation

Addition of serum from patients with chronic myeloid leukemia (CML) in both chronic phase and blastic transformation, to agar cultures of normal human marrow cells stimulated the growth of persisting colonies (day 35) containing either eosinophils or mast cells. Chronic phase serum stimulated an 800% increase in the total number of these colonies of which only 16% were mast cells. Serial studies using serum from 2 patients demonstrated that the proportion of mast cells increased during the progression to blastic transformation. The emergence of a greater proportion of persisting mast cell colonies and a decrease in absolute number of eosinophil colonies in agar cultures of normal marrow cells containing serum from patients with CML coincides with the emergence of blastic transformation and suggests that a significant change occurs in the absolute and relative concentration of hemopoietic growth factors in these patients.     

Characteristics of four new human cell lines derived from squamous cell carcinomas of the head and neck

Four human cell lines were established from biopsy specimens of squamous cell carcinomas of the larynx (TR131 and TR138), tongue (TR126), and buccal mucosa that had infiltrated a lymph node (TR146). All 4 lines readily formed colonies on a plastic substratum, but they were virtually incapable of forming colonies in an anchorage-independent semisolid support system of soft agar (cloning efficiencies, less than 0.02%). The proliferation of this group of tumor-derived cell lines, therefore, appeared to be highly anchorage dependent. Keratin filaments could be visualized in each line by indirect immunofluorescence with the use of polyclonal or monoclonal antibodies to keratins; staining with monospecific antibodies indicated that 3 of the 4 lines expressed simple epithelial keratins 8 and 18, whereas 1 of the 4 also expressed keratin 19. A panel of lectins revealed characteristic localization patterns distinct from those observed on other epithelial cell lines. Cells from 3 lines (TR131, TR138, and TR146) inoculated into nude mice (nu/nu) produced cystic nodules or unequivocal tumors having a histology indicating a squamous cell origin for the injected cells. Electron microscopy demonstrated that the cell lines covered a spectrum of differentiation capability ranging from the undifferentiated monolayer cultures of TR126 to the rather well differentiated, stratified cultures of TR131.     

In vitro growth of myeloid and erythroid progenitor cells from myelodysplastic patients in response to recombinant human granulocyte-macrophage colony-stimulating factor

Marrow progenitor cells from 14 myelodysplastic (MDS) patients and 17 normal donors were assayed in semisolid cultures supplemented with increasing doses of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or medium conditioned by 5637 bladder carcinoma cells (5637CM). At doses of supplements shown to be optimal for colony formation in cultures of normal marrow, myeloid (day 14) colony numbers were subnormal in 10 of 14 MDS marrows cultured in 5637CM and in 8 of 14 cultures containing rhGM-CSF (2.5 ng/ml). However, a high dose of rhGM-CSF (20 ng/ml) raised myeloid colony numbers in cultures of many MDS marrows, so that 9 of 14 now yielded colonies within the normal range; increased levels of 5637CM failed to do this. Erythroid colony growth was poor in 13 of 14 MDS marrow cultures supplemented with erythropoietin in addition to 5637CM or rhGM-CSF. High concentrations of rhGM-CSF did not increase erythroid growth. These data suggest that myeloid progenitors from the MDS clone may have a decreased responsiveness to hemopoietins which can be overcome at high concentrations of growth factors.