Carcinogenesis or tumourigenicity testing of animal cell lines for vaccine preparation by colony formation on soft agar and by agglutination under plant lectins

The carcinogenic or tumourigenic testing of seven animal kidney cell lines (F-81, CRFK, MDCK, Vero, Vero-2 cell line, MA-104 and BHK-21) established in China, were carried out in more than 700 nude mice for colony formation in soft agar and for agglutination under different density of plant lectins. Tests showed that there were correlation between cell line chromosome number variations and anchorage independence in soft agar, agglutinability under lectins and tumour-forming ability in nude mice. Since testing in vitro was more economical, simpler and faster and thus thought to be more reliable, we recommend measuring agglutinability, followed by anchorage independence or analysis of karyotype as the initial means for monitoring tumourigenicity of animal cell lines in nude mice.     

Tumorigenicity of green turtle fibropapilloma-derived fibroblast lines in immunodeficient mice

Fibroblast lines derived from normal skin and spontaneous or experimentally induced fibropapillomas of green turtles (Chelonia mydas) were established and propagated in medium composed of a combination of Dulbecco's minimal essential with F12 medium plus 10% fetal bovine serum at 30 degrees C. Fibropapilloma-derived fibroblasts were indistinguishable from normal skin fibroblasts in vitro. Tumor lines did not exhibit loss of contact inhibition, anchorage independence, or reduced serum requirements. Inoculation of primary and early-passage tumor cells into the medial margin of the pinna of C57BL/6J-nu/nu, C.B17-scid/scid, or NOD-scid/scid mice, however, resulted in fibroma formation, whereas inoculation of normal skin fibroblasts did not. Tumor-derived cells inoculated into the flanks of mice did not form tumors. The turtle origin of fibroblasts in tumors from mouse ears was confirmed by immunohistochemical and karyotype analysis. Fibroblast lines that were established from mouse ear fibromas had the normal karyotype (modal 2N = 55) of C. mydas. The cooler anatomic sites (ears) of immunodeficient mice are useful for confirming the tumorigenic (transformed) phenotype of green turtle fibropapillomatosis-derived fibroblasts. This mouse ear tumorigenicity test should facilitate studies of mechanisms of cellular transformation in green turtle fibropapillomatosis and other neoplastic diseases of poikilothermic vertebrates.     

Selenite-induced inhibition of colony formation by buthionine sulfoximine-sensitive and resistant cell lines

We previously demonstrated that treatment of HeLa cells with buthionine sulfoximine (BSO), which decreases the level of cellular glutathione, resulted in a decrease in the potency of selenite in inhibiting cell colony formation. We have now examined the effect of selenite on normal human lung fibroblast (CCL-210) cells, which resemble HeLa cells in their sensitivity to BSO, and on human lung adenocarcinoma (A549) cells, which are relatively insensitive to BSO. We have found that BSO treatment caused an approximately fourfold decrease in selenite potency in the CCL-210 cells, but had no significant effect on its potency in A549 cells. These results support the hypothesis that for selenite to exert its cytotoxic effect, it must under-go the reaction with an SH compound to form the selenotrisulfide. As a result of the lower sensitivity of the tumor cells to BSO, it was possible to achieve a large differential sensitivity to the cytotoxic effect of selenite.     

Tumorigenicity of persistently infected tumors in nude mice is a function of both virus and host cell type.

Although BHK-21 cells persistently infected with wild-type vesicular stomatitis virus (VSV) are sensitive to natural killer (NK) cells and do not form tumors in athymic nude mice, BHK-21 cells persistently infected with a previously isolated mutant virus (VSV-P) are resistant to NK cells and form tumors in nude mice. We used this VSV-P mutant to persistently infect HeLa cells and mouse tumor cell lines. A mouse mastocytoma line (P815) persistently infected with VSV-P was similar to BHK-21 cells in that it was resistant to NK cell lysis and formed tumors in nude mice. However, neither HeLa cells nor mouse myeloma lines persistently infected with VSV-P were resistant to NK cell lysis in vitro, and neither formed tumors in nude mice. Rejection by nude mice of HeLa cells and mouse myeloma cell lines persistently infected with VSV-P could be ablated by rabbit antiserum to asialo-GM1, implicating NK cells in the in vivo rejection of these persistently infected tumors. These results suggest that NK cell recognition and killing of virus-infected cells in vivo and in vitro depend upon genetic contributions from both the virus and the host cell.     

Tumorigenicity and H-2 expression of papillomavirus-transformed mouse cell lines

Summary Tumorigenicity in immunocompetent syngeneic mice and H-2 class I antigen expression of BPV1-transformed mouse cell lines had no correlation. H-2 expression was examined using monoclonal anti-(H-2K^b) and anti-(H-2D^b) antibodies in immunofluorescence staining for flow cytometry analysis and by determining the sensitivity of the cells to cytolysis by allostimulated spleen cells. Nontumorigenic cell lines were as resistant as tumorigenic cell lines to natural killer activity. The results indicate that in our model defence by natural killer cells is not a decisive factor. The results also show that instead of or in addition to H-2 class I antigens other factors (e. g. the presence or absence of virus-specific antigens) are important in determining the tumorigenicity of BPV1-transformed cell lines.     

Trichosanthin inhibits breast cancer cell proliferation in both cell lines and nude mice by promotion of apoptosis

Breast cancer ranks as a common and severe neoplasia in women with increasing incidence as well as high risk of metastasis and relapse. Translational and laboratory-based clinical investigations of new/novel drugs are in progress. Medicinal plants are rich sources of biologically active natural products for drug development. The 27-kDa trichosanthin (TCS) is a ribosome inactivating protein purified from tubers of the Chinese herbal plant Trichosanthes kirilowii Maximowicz (common name Tian Hua Fen). In this study, we extended the potential medicinal applications of TCS from HIV, ferticide, hydatidiform moles, invasive moles, to breast cancer. We found that TCS manifested anti-proliferative and apoptosis-inducing activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cells. Flow cytometric analysis disclosed that TCS induced cell cycle arrest. Further studies revealed that TCS-induced tumor cell apoptosis was attributed to activation of both caspase-8 and caspase-9 regulated pathways. The subsequent events including caspase-3 activation, and increased PARP cleavage. With regard to cell morphology, stereotypical apoptotic features were observed. Moreover, in comparison with control, TCS- treated nude mice bearing MDA-MB-231 xenograft tumors exhibited significantly reduced tumor volume and tumor weight, due to the potent effect of TCS on tumor cell apoptosis as determined by the increase of caspase-3 activation, PARP cleavage, and DNA fragmentation using immunohistochemistry. Considering the clinical efficacy and relative safety of TCS on other human diseases, this work opens up new therapeutic avenues for patients with estrogen-dependent and/or estrogen-independent breast cancers.     

In vitro traits of adenovirus-transformed cell lines and their relevance to tumorigenicity in nude mice

Six independently isolated adenovirus 2-transformed rat cell lines and one adenovirus 5-transformed human cell line have been examined in vitro for serum growth requirements, saturation density, anchorage-independent growth, proteolytic enzyme activity and the presence of LETS glycoprotein and T antigen. This series of adenovirus-transformed cell lines exhibits an oncogenic spectrum ranging from being tumorigenic in immunocompetent rats through to nontumorigenic in adult nude mice. The relevance of the in vitro findings to growth potential in vivo is discussed.     

Chemo-adoptive immunotherapy of nude mice implanted with human colorectal carcinoma and melanoma cell lines

Summary The antitumor effects of chemotherapy, recombinant human interleukin-2 (IL-2), recombinant human interferon A/D (IFN), allogeneic human lymphokine-activated killer (LAK) cells, and antitumor monoclonal antibody (mAb), administered alone and in various combinations, were tested in athymic nude mice carrying human tumor xenografts. Treatment began 6–18 days after i.v. or i.p. inoculation of colorectal carcinoma or melanoma cell lines, when macroscopic growths were evident. Chemotherapy consisted of two or three courses of 5-fluorouracil (5-FU) or dacarbazine. IL-2 and/or IFN were administered three to five times weekly for 1–3 weeks, usually starting 2–5 days after chemotherapy. Human LAK cells were infused once or twice weekly for 2 or 3 weeks concurrently with IL-2. In some experiments, murine anticolorectal carcinoma mAb (SF25) was administered. In both tumor systems, chemotherapy alone or immunotherapy alone (IL-2, IL-2 + LAK cells, IFN, IL-2 + IFN ± LAK cells) had little or no therapeutic effects. Additive effects were obtained by combining chemotherapy with IL-2 and LAK cells or with IL-2 and IFN. In the majority of the experiments, the most effective combination was chemotherapy + IL-2 + IFN + LAK cells. Treatment with mAb was beneficial in the colorectal carcinoma system when combined with 5-FU + IL-2 or 5-FU + IL-2 + IFN. Homing experiments with radiolabeled human and mouse LAK cells injected i.v. showed increased early accumulation in the liver and lungs, whereas freshly explanted mouse splenocytes localized mostly in the spleen and liver. The tissue distribution pattern of human LAK cells was similar in normal and tumor-bearing mice (with lung metastases). These findings suggest that combination of chemotherapy with cytokines and LAK cells can be partially effective for advanced solid human tumors even in the absence of the host's T-cell immune response. Preliminary experiments showed that tumor-specific, anti-melanoma T-cell clones were effective in local (s.c.) tumor growth inhibition (Winn assay) following coinjection with the autologous tumor cells.     

Establishment and characterization of cell lines derived from nude mice transplanted squamous cell carcinoma of uterine cervix

Two human cell lines, KIMI-1 and -2 were established from nude mice transplanted tumor originated from a human squamous cell carcinoma of the uterine cervix. These two cell lines have different shapes, chromosome numbers and tumor markers, respectively.     

Effects of hormonal growth factors on colony formation and chemosensitivity of human tumors in agarose

Summary Because low plating efficiencies of most human cancers severely limit the number of successful chemosensitivity tests that can be performed, we studied the growth-enhancing effects of hormonal growth factors on a variety of solid tumors. Dose-response studies with progesterone and estradiol indicated no benefit from adding these substances to the culture medium. This was true whether progesterone or estradiol was used alone or in the presence of other hormones. By contrast, epidermal growth factor (EGF) at concentrations from 10 to 100 ng/ml increased colony numbers up to 10-fold. Although insulin, hydrocortisone, and EGF used alone could either stimulate or inhibit the growth of specific tumors, the combination of all three (hormone mixture or HM) was always at least as good and usually better than any individual component in increasing cloning efficiency. HM-supplemented medium gave significantly increased colony counts in 41/46 tumors. Sensitivity to anticancer drugs was not changed in 63 paired drug tests.     

Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions

The aim of this study was to compare the in vitro effects of glial cell line-derived neurotrophic factor, stem cell factor, granulocyte macrophage-colony stimulating factor, and co-culture with Sertoli cells on the efficiency of adult mouse spermatogonial stem cells colony formation. For these purpose, both Sertoli and spermatogonial cells were isolated from adult mouse testes. The identity of the cells was confirmed through analysis of alkaline phosphatase activity, immunocytochemistry against OCT-4, c-kit, and vimentin, and also by transplantation of these cells in the recipient testes. The isolated spermatogonial cells were treated either with various concentrations of the above mentioned factors or co-cultured with Sertoli cells for 3 wk. The spermatogonial cells of the resulting colonies were transplanted via rete testis into the mouse testes, which were irradiated with 14 Gy. The results indicated that glial cell line-derived neurotrophic factor is the most appropriate factor for in vitro colonization of adult mice spermatogonial cells compared with other cytokines and growth factors. A short-term co-culture with Sertoli cells showed a significant increase in the number and diameter of the colonies compared with the treated growth factors and the control group. We have also demonstrated that mouse spermatogonial stem cells in the colonies after co-culturing with Sertoli cells could induce spermatogenesis in the recipient testes after transplantation.     

The effect of culture conditions on colony morphology and proliferative capacity in human prostate cancer cell lines

Primary cultures and cell lines form three types of colonies, termed holoclones, meroclones and paraclones by Barrandon and Green (Proc Natl Acad Sci U S A 84:2302-2306, 1987). They suggested that the three types correspond to colonies derived from stem, transit-amplifying and terminally differentiated cells. We determined the effect of culture conditions (seeding density, serum concentration, type of medium and substrate) on the proportion of each colony type and the cell number of individual colonies, using three prostate cancer cell lines, DU145, LNCaP and PC-3. In less favourable culture conditions, stem cell (SC) colonies tended to be lost; but in more favourable conditions, only modest increases in the proportion of SC colonies were observed. Under some conditions, cell number, but not colony-forming ability, was altered, indicating that colony cell number is controlled, at least in part, by different factors to colony formation. Colony-forming ability of individual cell lines is remarkably stable and there is little evidence for clonal evolution in culture, which might be expected and would result in more aggressive, faster-growing cells. Better understanding of how colony-forming efficiency is controlled could lead to the identification of drug targets that control SC growth and modify the progression of cancer.     

Establishment and characteristics of Gottingen minipig skin in organ culture and monolayer cell culture: relevance to drug safety testing

Skin from Gottingen minipigs was used as a source of tissue for organ and cell culture and compared to human skin for growth conditions and sensitivity to irritants. Optimal organ culture conditions were determined, based on the preservation of the histological structure. These included serum-free, growth factor-free conditions with a calcium concentration of 1.5mM. Formulations in which the calcium concentration were low (0.075-0.15mM) failed to support tissue viability (even in the presence of dialyzed serum). Epidermal keratinocytes were grown from tissue explants and as single cells from enzyme-disrupted tissue. Optimal keratinocyte growth was achieved using a serum-free, growth factor-supplemented culture medium with a calcium concentration of 0.15mM. Fibroblasts were optimally grown from explant cultures using a medium with 1.5mM calcium and 10% fetal bovine serum. The conditions that were optimal for maintenance of intact pig skin, as well as for the isolated cells, are the same conditions that have been shown previously to be optimal for intact human skin and skin cells. In additional studies, pig skin keratinocytes and fibroblasts were exposed to a panel of contact irritants and contact sensitizers. Using growth inhibition as the response, the median effective dose values with each agent were very similar to the values previously determined for human epidermal keratinocytes and human dermal fibroblasts. Taken together, these data suggest that the skin from the Gottingen minipig can be used as a surrogate for human skin in ex vivo skin safety studies.     

Establishment of human colon carcinoma lines in nude mice.

After subcutaneous inoculation into nude mice of 24 human colon adenocarcinomas, growth, defined as histopathologically confirmed tumor growth which has been passed, was observed in 13 cases (54%). Tumors from metastatic sites showed higher take rates (58%) than tumors from primary sites or recurrent tumors (50%). Nine continuous tumor lines were established (69% of growing tumors) with metastatic tumors establishing more readily (100% of growing tumors) than primary tumors (40%). The average period in primary transplant was shorter for metastasis (8.3 weeks), than for primary tumors (18.5 weeks); total material 10.6 weeks. Average periods between passages were shorter than primary transplant times; these periods were shorter for metastases (6.6 weeks) than for primary tumors (9.4 weeks); total material 7.4 weeks. Of four growing tumors not established as continuous lines, three were primary and one a recurrent tumor, and the loss of tumor growth occurred in very early passages, not later than passage 3. All nude mouse-grown colon tumors were moderately well differentiated.     

Optimization of human skeletal muscle precursor cell culture and myofiber formation in vitro

Muscle bioengineering is proposed as a treatment option for various conditions requiring restoration of muscle function. In order to allow for rapid clinical translation culture conditions have to be optimized for human application. The optimal isolation and culture technique should be able to support cell growth and differentiation using defined media only. Therefore, we have evaluated alternative culture conditions to determine the optimal growth condition for the engineering of human skeletal muscle. In this research, we present protocols for consistent isolation and growth of human muscle precursor cells (MPCs). MPCs were grown from human biopsies and expanded in culture using defined media and collagen coated dishes only. The best results were achieved using a one-step pre-plating and by supplementing the growth medium with insulin, dexamethasone, human basic fibroblast growth factor (hFGF) and human epithelial growth factor (hEGF). Detailed cell characterization using fluorescence-activated cell-sorting analysis and morphological analysis at different passages were performed. Further, the applicability of these cells for tissue engineering purposes was assessed by measuring expansion potential, formation of myofibers and fused myotubes. We have established a culture technique for human MPCs that allows for reliable cell growth and expansion using collagen coated dishes and defined media only. Cell characterization demonstrated a muscle phenotype and the ability to form myofibers in vitro.     

Effects of bacterial agarase on agarose gel in cell culture

Summary Bacterial agarase, concentrated and purified from culture filtrate of agar-degrading bacteria, has been used to clean cells cultured in soft agarose from gel residues. The enzyme also has been used to liquefy the gel directly in the dishes to facilitate the removal of cells. The surfaces of glioma cells from agarase-treated colonies could not be distinguished in the scanning electron microscope from surfaces of cells which had never been in contact with agarose or agarase. This implies that most agarose residues had been removed, and also that the treatment did not seriously alter the cell surfaces. The influence of the agarase treatment also was tested by comparison of the mitotic index and the incorporation of [³H]thymidine in agarase-treated and untreated cells. No effects of the treatment could be seen in these tests. The work has been supported by the Swedish Cancer Society and the Swedish Natural Science Research Council.