Establishment of normal diploid and malignant heteroploid cell lines from non-treated and benzo(a)pyrene treated hamster embryo cell cultures

Summary Two normal diploid control cell lines and a heteroploid malignant transformed cell line from B(a) P treated hamster embryo cell cultures were established. The 14-month-old B(a)P transformed cell line grew 8-times faster than the 20-month-old control cell line. The control cell line showed normal diploid chromosome complement in 93% cells and heteroploidy in 7% cells while B(a)P treated line showed 83% heteroploid cells and only 17% diploid cells. This is the first report on the establishment of diploid hamster cell cultures grown for extended period. Acknowledgments. This work was supported by the French Ministry of Quality of Life, by the Institut National de la Santé et Recherche Médicale, by the S.E.I.T.A. and by Contract No. 12-14-7001-297 of the Agricultural Research Service, U.S. Dept. of Agriculture, Administered by the Athens, Georgia Area, Richard B. Russell Agricultural Research Center, Athens, Georgia 30604, USA. We thank Mr.B. R. Sharma, Mr.E. J. Radin, Dr.R. Hakim and Dr.Ved Brat for their help during this work.     

Mitogenic effect of the serum of rats exposed to cold

Summary Serum from rats exposed for 3 days to 5 °C stimulated the multiplication of mouse heteroploid and human and rat diploid cells in culture. The response of the cells to this unspecific growth stimulation was related to the growth capacities of the different cell types.     

Silencing of RB1 but not of RB2/P130 induces cellular senescence and impairs the differentiation potential of human mesenchymal stem cells

Stem cell senescence is considered deleterious because it may impair tissue renewal and function. On the other hand, senescence may arrest the uncontrolled growth of transformed stem cells and protect organisms from cancer. This double function of senescence is strictly linked to the activity of genes that the control cell cycle such as the retinoblastoma proteins RB1, RB2/P130, and P107. We took advantage of the RNA interference technique to analyze the role of these proteins in the biology of mesenchymal stem cells (MSC). Cells lacking RB1 were prone to DNA damage. They showed elevated levels of p53 and p21^cip1 and increased regulation of RB2/P130 and P107 expression. These cells gradually adopted a senescent phenotype with impairment of self-renewal properties. No significant modification of cell growth was observed as it occurs in other cell types or systems. In cells with silenced RB2/P130, we detected a reduction of DNA damage along with a higher proliferation rate, an increase in clonogenic ability, and the diminution of apoptosis and senescence. Cells with silenced RB2/P130 were cultivated for extended periods of time without adopting a transformed phenotype. Of note, acute lowering of P107 did not induce relevant changes in the in vitro behavior of MSC. We also analyzed cell commitment and the osteo-chondro-adipogenic differentiation process of clones derived by MSC cultures. In all clones obtained from cells with silenced retinoblastoma genes, we observed a reduction in the ability to differentiate compared with the control clones. In summary, our data show evidence that the silencing of the expression of RB1 or RB2/P130 is not compensated by other gene family members, and this profoundly affects MSC functions.     

Interferon-beta can induce the production of plasminogen activator by cultured human cancer cells

Three cultured human cell lines, renal cancer cells (ACHN), bladder cancer cells (EJ), and fibroblasts transformed in culture by Co-60 gamma rays (KMST-6), when treated with interferon-beta, produced 1.5 to 4 times as much plasminogen activator as the untreated control cultures. This enhanced production of PA was inhibited by cycloheximide or actinomycin D.     

The use of 5-fluorocytosine and ketoconazole in the culture of the erythrocytic stages of Plasmodium falciparum and some tumor cell lines

In vitro culture systems are often contaminated by bacteria and fungi. It is therefore often necessary to supplement culture media with agents such as penicillin/streptomycin, gentamycin or amphotericin B. The latter cannot be used in the in vitro culture of erythrocytic stages of P. falciparum, and thus anti-fungal agents have not been regularly used in this system. We describe the prophylactic use of 5-fluorocytosine (5-FC) and ketoconazole (KTZ) in tissue cultures at concentrations up to 300 and 10 micrograms/ml respectively which have no effect on the growth of P. falciparum (FCR-3 strain). A melanoma cell line (C32) and a line of uterine carcinoma (C41) were also unaffected by similar concentrations of 5-FC and KTZ. When dissolved in complete culture medium (RPMI 1640) with 10% human plasma, the minimum inhibitory concentration of 5-FC for a susceptible strain of Candida remained below 2 micrograms/ml. These experiments suggest that 5-FC (at 50 micrograms/ml) alone or in combination with KTZ (at 1 microgram/ml) is a useful addition to the armamentarium of antimicrobials available to the tissue culture biologist for a variety of cell culture systems.     

肝癌细胞系Hep3B中CD133+细胞亚群的分离及其特性的研究

目的研究CD133在人肝癌细胞系Hep3B中的表达以及CD133+细胞的体外增殖、自我更新及体内成瘤能力,初步探讨肝癌中CD133+细胞亚群的干细胞特性。方法流式细胞仪检测未分选的Hep3B细胞中CD133+细胞表达情况;免疫磁珠分选技术纯化CD133+肿瘤细胞;MTT法检测CD133+细胞体外增殖能力;无血清培养纯化...     

Transformation, in vitro, of cerebral hamster cells by polyoma virus]

A cell line called HCxPy was obtained in vitro by transformation of dissociated hamster brain cell cultures by polyoma virus. The first foci of transformed cells became evident 90 to 120 days after viral infection. This cell line is now at the 46th passage. The cells appear tumorigenic for hamsters after subcutaneous and intracerebral injection. They carry the polyoma virus T and cell surface antigens. Good evidence for astrocytic differentiation can be found by morphological examination of the tumours and of the cultured cells.     

Quantitative studies on in vitro transformation of hamster chondrocytes.

Hamster chondrocytes could be transformed in a quantitative assay system which used X-irradiated feeder layer cells. Morphological transformation occurred on addition of, 4NQO, but not in control cultures. Differentiation was classified into 3 types (good, poor and none); normal and transformed colonies contained similar proportions of the 3 types.     

Long-term production and culture of clones of human T-lymphocytes

Culture of blood T lymphocytes collected from normal individuals and cancer patients were carried out in presence of T cell growth factor (TCGF); these cultures presented cytotoxic activity directed against different targets (lectin activated cells, autologous cancer cells, antibody coated cells and K 562). In order to study separately the different effector subpopulations, isolation of single cultured cells were performed with the help of a micropipette under microscope and monoclonal cultures were carried out in presence of TCGF. In the preliminary cytotoxic assays performed in the clones: (1) a marked activity directed against lectin targets was observed in many clones and (2) an important N K activity was exhibited by the clone 45 B9 (65% of the tested cells lysed human lymphoma K 562 cells).     

Interferon-β can induce the production of plasminogen activator by cultured human cancer cells

Summary Three cultured human cell lines, renal cancer cells (ACHN), bladder cancer cells (EJ), and fibroblasts transformed in culture by Co-60 gamma rays (KMST-6), when treated with interferon-, produced 1.5 to 4 times as much plasminogen activator as the untreated control cultures. This enhanced production of PA was inhibited by cycloheximide or actinomycin D.     

Heat evolution of cultured human keratinocytes

Summary The heat production of normal and transformed human epidermal keratinocytes precultured in Petriperm ^tm tissue culture dishes was measured calorimetrically. For this purpose, the membrane at the bottom of the culture dish was cut out aseptically and put into a microcalorimeter vessel with the cell layer inwards. A continuous heat output of (83±12) pW/cell was measured for normal keratinocytes from a confluent primary culture. A value of (134±35) pW/cell was obtained when the transformed kerationocyte line SV-K14 was used. The method described in this paper is simple, leads to reproducible results, and can be easily adapted to the calorimetric study of other mammalian cells in vitro.     

Presence of oncornavirus-like particles in the P388 murine leukemic cell line

A culture of P388 murine lymphoblastoid cells has been shown to contain type C oncornavirus-like particles budding at the plasma membrane. Occasionally intracytoplasmic type A and immature type B particles were also observed by electron microscope techniques. The discovery of oncornavirus-like particles in the P388 cell line increases the utility of this neoplastic system for detecting potential antineoplastic agents.     

Oxidative modifications of low-density lipoproteins (LDL) by the human endothelial cell line EA.hy 926

Modifications of LDL by the EA.hy 926 cell line were compared to those generated by human umbilical vein endothelial cells (HUVEC). Thiobarbituric acid reactive substances (TBARS) index values (TBARS sample/TBARS cell-free control ratio) were 2.64±0.18 (m±SE, n=11) and 3.12±0.24 (n=11), for HUVEC and EA.hy 926, respectively. The percentage of the most electronegative modified LDL fraction (fraction C), assessed by using an ion-exchange chromatographic method based on fast protein liquid chromatography (FPLC), represented 14±3% (n=34) and 22±13% (n=10) of total modified LDL in HUVEC and EA.hy 926, respectively. LDL modified by both cell lines showed increased agarose electrophoretic mobility and apo B100 fragmentation on SDS-PAGE. None of the results were significantly different between the two cell lines. Superoxide anion production was 0.12±0.04 (n=11) and 0.07±0.01 nmol/min/mg cell protein (n=11) in HUVEC and EA.hy 926, respectively. Cell-specific effects on LDL were abrogated in cysteine-free medium. Moreover, cell-modified LDL were similarly degraded by J774 macrophage-like cells. We conclude that EA.hy 926 cells are a good model for investigating endothelial cell-induced modifications of LDL. Advantages include ready availability and less individual variability than with HUVEC.     

Dexamethasone enhances CTLA-4 expression during T cell activation

T cell activation is enhanced by the costimulatory interaction of B7 on antigen-presenting cells and CD28 on T cells, resulting in long-term T cell proliferation, differentiation and production of large amounts of cytokines, such as interleukin (IL)-2. CTLA-4 is a co-stimulation receptor that shares 31% homology with CD28 and binds B7 family members with higher affinity. CTLA-4 is transiently expressed intracellularly and on the cell surface following activation of T cells. We have studied the kinetics of CTLA-4 expression and the effects of dexamethasone on CTLA-4 expression during T cell activation in cultures of mouse spleen cells stimulated by a mixture of immobilized anti-CD3 and anti-CD28 monoclonal antibodies (anti-CD3/CD28 mAb) or concanavalin A (ConA). CTLA-4 expression peaked on day 2 and returned to background levels after 7 days. Dexamethasone was found to potentiate CTLA-4 expression in a dose-dependent manner with an EC₅₀ effective concentration 50%) of about 10⁻⁸ M. In contrast, other immunosuppressive agents, such as rapamycin or cyclosporin A had no or an inhibitory effect on CTLA-4 expression, respectively. Dexamethasone also stimulated CD28 expression, but inhibited IL-2R expression during anti-CD3/CD28 mAb-induced mouse splenic T cell activation. Western blot analyses of lysates of activated mouse T cells showed that dexamethasone increased CTLA-4 protein levels twofold during anti-CD3/CD28 mAb-induced activation. Dexamethasone also enhanced CTLA-4 messenger RNA twofold as quantified by ribonuclease protection assay. The effects of dexamethasone on CTLA-4 expression were glucocorticoid-specific and completely inhibited by the glucocorticoid receptor antagonist mifepristone (RU486), indicating that the effect of dexamethasone on CTLA-4 expression is mediated through the glucocorticoid receptor. In conclusion, the immunosuppressive agent dexamethasone actually stimulates CTLA-4 expression, which is involved in downregulation of T cell activation. Received 19 May 1999; received after revision 13 July 1999; accepted 13 July 1999     

Tissue antagonist of interferon : murine substance similar to plant lectins]

A tissue antagonist of interferon (TAI) extracted from mouse costal cartilage contains a substance which has many properties characteristic of plant lectins. After binding to the cell membrane receptors, it agglutines normal and transformed murine cells. In interferon treated cells, it restores virus sensitivity probably through a modification in the distribution of membrane bound cellular antigens.     

Quantitative studies on in vitro transformation of hamster chondrocytes

Summary Hamster chondrocytes could be transformed in a quantitative assay system which used X-irradiated feeder layer cells. Morphological transformation occurred on addition of, 4NQO, but not in control cultures. Differentiation was classified into 3 types (good, poor and none); normal and transformed colonies contained similar proportions of the 3 types.This work was supported by a grant from the Ministry of Education, Science and Culture, Japan.We thank Miss M. Tanaka for her technical assistance.     

Impact of interactions between normal and transformed epithelial cells and the relevance to cancer

The majority of human cancers are initiated when a single cell in an epithelial sheet becomes transformed. Cell transformation arises from the activation of oncoproteins and/or inactivation of tumor suppressor proteins. Recent studies have independently revealed that interaction and communication between transformed cells and their normal neighbors have a significant impact on the fate of the transformed cell. Several reports have shown that various phenomena occur at the interface between normal and transformed epithelial cells following the initial transformation event. In epithelia of Drosophila melanogaster, transformed and normal cells compete for survival in a process termed cell competition. This review will summarize current research and discuss the impact of these studies on our understanding of how primary tumors emerge and develop within a normal epithelium.     

Rethinking synchronization of mammalian cells for cell cycle analysis

An analysis of different classes of forced or batch synchronization methods reveals why these methods, in theory, do not produce synchronized cultures. Cells may be aligned for a particular property after specific treatments, but these aligned cells do not correspond to any particular cell age during the normal cell cycle. The experimental methods analyzed are those that arrest cells with a G1 phase amount of DNA, those that inhibit DNA synthesis, and those that arrest cells at mitosis. Release of arrested cells from inhibition does not produce cells reflecting cells during the normal division cycle. Thus, cells produced by batch or forcing methods are not experimental models for analysis of the normal cell cycle.     

The use of 5-fluorocytosine and ketoconazole in the culture of the erythrocytic stages ofPlasmodium falciparum and some tumor cell lines

Summary In vitro culture systems are often contaminated by bacteria and fungi. It is therefore often necessary to supplement culture media with agents such as penicillin/streptomycin, gentamycin or amphotericin B. The latter cannot be used in the in vitro culture of erythrocytic stages ofP. falciparum, and thus anti-fungal agents have not been regularly used in this system. We describe the prophylactic use of 5-fluorocytosine (5-FC) and ketoconazole (KTZ) in tissue cultures at concentrations up to 300 and 10 g/ml respectively which have no effect on the growth ofP. falciparum (FCR-3 strain). A melanoma cell line (C32) and a line of uterine carcinoma (C41) were also unaffected by similar concentrations of 5-FC and KTZ. When dissolved in complete culture medium (RPMI 1640) with 10% human plasma, the minimum inhibitory concentration of 5-FC for a susceptible strain ofCandida remained below 2 g/ml. These experiments suggest that 5-FC (at 50 g/ml) alone or in combination with KTZ (at 1 g/ml) is a useful addition to the armamentarium of antimicrobials available to the tissue culture biologist for a variety of cell culture systems.     

Karyological studies on established mosquito cell lines

Chromosome frequency distribution and cellular DNA estimations in different established mosquito cell lines were studied. These cell lines exhibited a wide range of cell types with a diploid stem-line comprising 50-55% and a haploid substem-line comprising 12-30% of the population. Estimation of cellular DNA contents by impulse cytoflowmetry and by Feulgen cytophotometry supported these observations. Because of their low diploid counts, these cell lines cannot be classified as diploid.