Development of intestinal epithelial cell lines using a transgenic mouse

The epithelial cells of the colonic mucosa of the animal have proved impossible to culture using standard tissue culture techniques. Immortalization of adult colonic epithelial cells has been unsuccessful due to the lack of DNA synthesis in these cells once they are isolated from the tissue. Recently an unique transgenic mouse bearing a temperature sensitive mutant of the known immortalizing gene, SV40 large T has become available. The advantage of this mouse is that the SV40 large T gene is expressed in every cell. Active immortalizing protein is produced in each cell at the permissive temperature. We have used colonic mucosa from these mice to initiate cultures of epithelial cells from the colon of adult mice. The cells grow readily at the permissive temperature but die within 7 days at the non-permissive temperature. The methods used to develop these cultures are described.     

Why Are Human Cells Resistant to Malignant Cell Transformation in vitro?1

Transformation of human cells, both induced and spontaneous, is an extremely rare event, whereas rodent cells are relatively easily transformed when treated with a single carcinogenic agent. The present review addresses the question of why human cells are resistant to malignant transformation in vitro. To facilitate understanding of the problem, the process of transformation is divided operationally into two phases, i.e. phase I, immortalization; and phase II, malignant transformation. In human cells, one-phase transformation, i.e., the consecutive occurrence of phases I and II due to the action of a single carcinogenic agent, is observed only rarely. Once human cells are immortalized, however, malignant transformation by chemical carcinogens or oncogenes proceeds, suggesting that for human cells, phase I immortalization is a prerequisite for such transformation to take place. To date, about 20 papers have been published describing protocols for the two-phase transformation of a variety of human epithelial cells and fibroblasts. In most experiments, SV40, human papilloma viruses and their transforming genes are utilized for induction of phase I (immortalization) followed by the use of chemical carcinogens or activated oncogenes for induction of phase II (malignant transformation). Possible mechanisms that would render human cells refractory to transformation are discussed below.     

Treatment with aflatoxin B1 for immortalization of human fibroblasts from Li-Fraumeni patients

Immortalization of normal human fibroblasts is a very rare event. Multiple genes such as p53 and cellular senescence genes are possibly involved in immortalization of human fibroblasts, suggesting that multiple treatments with carcinogens are required for the immortalization. We describe here the procedure for immortalization of human fibroblasts (MDAH 087) from Li-Fraumeni cancer syndrome with a germ-line p53 mutation. The cells were subjected to multiple treatments with aflatoxin B₁ (AFB₁) in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), and 3 of 9 MDAH 087 cell cultures treated 1–3 times with 0.1–1 µg/ml AFB₁ became immortal, defined as continuous growth for over 300 population doublings after the first treatment. However, cultures of human fibroblasts from a normal embryo treated under the same conditions failed to escape senescence. The results indicate that the model of human fibroblasts with a mutated p53 allele exposed to AFB₁ is potentially useful for studying mechanisms of chemically induced immortalization.     

In vitro negative selection of αβ T cell receptor transgenic thymocytes by conditionally immortalized thymic cortical epithelial cell lines and dendritic cells

We have established conditionally immortalized thymic cortical epithelial cell lines from transgenic mice carrying a temperature-sensitive SV40 large Tantigen. One of these cell lines expresses cortical markers and produces IL-1α, IL-6, IL-7, and TGF-β1. These cells express class I major histocompatibility complex (MHC) constitutively and class II MHC upon induction with IFN-μ. The cells appear to have a normal class I antigen presenting pathway since messages for both peptide transporter genes (TAP1, TAP2) were detected. The ability of these cortical epithelial cells to present peptide antigen was compared to that of thymic dendritic cells. In suspension culture with αβ Tcell receptor (TcR) transgenic thymocytes, these epithelial cells and dendritic cells (pre-pulsed with peptide cognate for the transgenic TcR) caused down-regulation of CD4, CD8, and TcR in an antigen dose-dependent and MHC-restricted manner. CD4^dullCD8^dull cells were taken as evidence for negative selection because these cells contained apoptotic DNA. Concentration of peptide required for negative selection of thymocytes was similar between dendritic cells and cortical epithelial cells. In contrast, αβ transgenic spleen cells were activated only by dendritic cells but not by cortical epithelial cells.     

Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes

BACKGROUND/AIMS: Liver failure is a life threatening condition currently treated by palliative measures and, when applicable, organ transplantation. The use of a bioartificial organ capable of fulfilling the main functions of the liver would represent an attractive alternative. However, the shortage of suitable donor cells, and their limited growth ability have impeded the development of this strategy. We investigated whether lentiviral vectors allow for conditional immortalization of human hepatocytes and whether these immortalized hepatocytes could reverse lethal acute liver failure. METHODS: We exposed primary human hepatocytes to Cre-excisable lentiviral vectors coding for SV40T Antigen, telomerase, and/or Bmi-1 and tested the functionality of the resulting cell lines. Therapeutic potential of immortalized hepatocytes were tested in a murine model of acetaminophen-induced hepatic injury. RESULTS: The immortalized hepatocytes grew continuously yet were non-tumorigenic, stopped proliferating when exposed to Cre recombinase, and conserved defining properties of primary hepatocytes, including the ability to secrete liver-specific proteins and to detoxify drugs. The implantation of encapsulated immortalized human hepatocytes rescued mice from lethal doses of acetaminophen. CONCLUSIONS: Lentiviral vectors represent tools of choice for immortalization of non-dividing primary cells, and lentivirally immortalized human hepatocytes are promising reagents for cell-based therapy of acute liver failure.     

Genetic Complementation of the Immortal Phenotype in Group D Cell Lines by Introduction of Chromosome 7

Human immortal cell lines have been classified into at least four (A–D) genetic complementation groups by cell-cell hybrid analysis, i.e., a hybrid derived from different groups becomes mortal. Recently we have demonstrated that introduction of human chromosome 7 suppresses indefinite division potential in the non-tumorigenic human immortalized fibroblast lines KMST-6 and SUSM-1, both assigned to complementation group D. By extending our microcell-mediated chromosome transfer, we found that chromosome 7 also suppresses division potential in the human hepatoma line HepG2 (again, assigned to group D). Chromosome 7 was thus shown to suppress indefinite growth in the above group D cell lines irrespective of their cell types, or whether they are tumorigenic or not. Since chromosome 7 had no such effect on representative cell lines derived from complementation group A, B or C, these results indicate that the senescence gene(s) commonly mutated in the group D cell lines is located on chromosome 7.     

人卵巢癌永生化细胞系BUPH:OVSC-2的建立及其生物学特性研究

背景与目的：利用永生化技术建立预期表型的细胞系,已成为建设系的重要手段之一,而人卵巢癌永生化细胞系的建立国内外少有报道。本文介绍人卵巢肉瘤样癌永生化细胞系的建立,以及对其生物学特性的研究。方法：以手术切除的卵巢肉瘤样癌腹壁转移组织为材料,进行体外培养。将永生化基因SV40T抗原基因转染第10代细胞,经筛选、抗性克隆扩大培养,得到永生化细胞系。通过光学显微镜、电子显微镜、生化曲线测定、染色体分析、双层软琼脂培养、裸鼠接种、免疫组化等,研究其生物学特性,并与其来源细胞的生物学特性进行比较。结果：建立一株人卵巢肉瘤样癌永生化细胞系,命名为BUPH：OVSC－2,现已传至90年代。其生物学特性为：形态学观察细胞呈肉瘤样细胞形态,超微结构证实为上皮起源;细胞生长旺盛;具有恶性细胞的特征,恶性度高。通过比较,与其来源细胞的生物学特性无明显差别。结论：BUPH：OVSC－2为一株恶性度高的人卵巢肉瘤样癌永生化细胞系,保留了其来源细胞的生物学特性,可作为卵巢肉瘤样癌研究的实验模型。     

Living forever and dying in the attempt

Since the first cell culture was set at the beginning of the twentieth century it was believed that all cultured cells, if provided with the proper conditions, would replicate indefinitely. Sixty years later we overthrew this dogma by finding that normal cells have a finite capacity to replicate and that only abnormal or cancer cell populations can replicate indefinitely. We interpreted these findings to bear on our understanding of the aging process. If, as had been previously thought, normal cells can replicate indefinitely, then age changes could not have an intracellular origin. Our findings demonstrated that, on the contrary, age changes do have an intracellular origin. The hundreds of changes that were subsequently found to precede the loss of replicative capacity have been interpreted to be age changes and the finitude of replication to be an expression of longevity determination. Subsequent findings by others have determined the molecular mechanism that governs the finitude of normal cell replicative capacity and how immortal cancer cells escape this inevitability. Thus, key events in our understanding of aging, longevity determination and cancer have been revealed.