Tumor-derived Human Cell Lines Research Articles

Immortal, telomerase-negative cell lines derived from a Li-Fraumeni syndrome patient exhibit telomere length variability and chromosomal and minisatellite instabilities.

Five immortal cell lines derived from a Li-Fraumeni syndrome patient (MDAH 087) with a germline mutant p53 allele were characterized with respect to telomere length and genomic instability. The remaining wild-type p53 allele is lost in the cell lines. Telomerase activity was undetectable in all immortal cell lines. Five subclones of each cell line and five re-subclones of each of the subclones also showed undetectable telomerase activity. All five immortal cell lines exhibited variability in the mean length of terminal restriction fragments (TRFs). Subclones of each cell line, and re-subclones of the subclones also showed TRF variability, indicating that the variability is owing to clonal heterogeneity. Chromosome aberrations were observed at high frequencies in these cell lines including the subclones and re-subclones, and the principal types of aberrations were breaks, double minute chromosomes and dicentric chromosomes. In addition, minisatellite instability detected by DNA fingerprints was observed in the immortal cell lines. However, all of the cell lines were negative for microsatellite instability. As minisatellite sequences are considered recombinogenic in mammalian cells, these results suggest that recombination rates can be increased in these cell lines. Tumor-derived human cell lines, HT1080 cells and HeLa cells that also lack p53 function, exhibited little genomic instability involving chromosomal and minisatellite instabilities, indicating that chromosomal and minisatellite instabilities observed in the immortal cell lines lacking telomerase activity could not result from loss of p53 function.

Volatile organic compounds cytotoxicity and expression of HSP72, HSP90 and GRP78 stress proteins in cultured human cells

The aim of this study was to determine whether overexpression of stress proteins (SPs) could be a sensitive biomarker for cell injury due to exposure to low doses of volatile organic compounds (VOCs) such as benzene, ethylbenzene, toluene, xylene, and chlorinated derivatives (ClB). Sublethal and cytotoxic threshold concentrations of the VOCs were determined by studying the growth rate of normal (fibroblasts) or tumor-derived human cell lines (A549, HepG2) exposed for 4 days to VOCs. Changes in SP expression as a function of concentrations were investigated by Western blotting. VOC toxicity was found to be correlated with their degree of chlorination and their hydrophobicity. Cytotoxic threshold concentrations (no-observed effect concentration, NOEC) were found to be similar for the three cell lines. It was observed that using a mixture of VOCs, each of them at concentration below the NOEC, resulted in an actual toxicity to the cells. This finding reveals a synergistic effect and should be taken into account when assessing threshold risk and exposure limit values in the worker's environment when several pollutants may be present. HSP72 and HSP90 expression levels were not affected whereas GRP78 expression was increased by all the VOCs. Taking into account the specific molecular function of GRP78, it suggests that VOC exposure results in misfolded or underglycosylated protein accumulation in the endoplasmic reticulum. GRP78 overexpression was closely related to the magnitude of growth inhibition due to increasing concentrations of each VOC. The overexpression was found to be significant for concentrations 5 to 30 times higher than NOEC, indicating that, under our experimental conditions, GRP78 expression cannot be considered as a sensitive biomarker of exposure to environmental VOCs.

Up-regulation of CIR1/CROC1 expression upon cell immortalization and in tumor-derived human cell lines.

Acquisition of the immortal phenotype by tumor cells represents an essential and potentially rate-limiting step in tumorigenesis. To identify changes in gene expression that are associated with the early stages of cell immortalization, we compared genetically matched pairs of pre-immortal and immortal human cell clones by mRNA differential display. Two transcripts, denoted CIR1 and CIR2, were identified which were up-regulated in immortal cells. Sequence analysis revealed CIR1 to be identical to the recently cloned CROC1/UEV-1 gene, whereas CIR2 corresponds to an as yet uncharacterized 1.2 kb mRNA. A 5-6-fold elevation in CIR1/CROC1 expression and a 2-3-fold elevation in CIR2 expression were observed in SV40-transformed human embryonic kidney cells immediately following proliferative crisis, suggesting a potential role for these genes in immortalization. Expression of CIR1/CROC1 was found to be elevated also in a variety of immortal human tumor-derived cell lines, as compared to their normal tissue counterparts. These results are compatible with induction of CIR1/CROC1 being an early event in the acquisition of immortality and with a role for this gene in the immortal phenotype of tumor cells.

A role for the interferon-inducible protein 10 in inhibition of angiogenesis by interleukin-12.

We have developed a nude mouse model in which tumor regression is reproducibly induced by coinjection with or intratumor inoculation of EBV-immortalized B cells. A wide spectrum of tumor-derived human cell lines can be established as subcutaneous tumors in sublethally irradiated athymic mice. Most of these tumors are induced to regress by human B cells immortalized with EBV. The tumor regression process is characterized by superficial necrosis and scarring that progressively extends itself to involve all or most of the tumor. Microscopically, tumor regression is characterized by tumor tissue necrosis, evidence of vascular damage with intimal thickening and capillary thrombosis, and macrophage, but not lymphocyte or neutrophil, infiltration. Profiles of cytokine expression differed between progressive and regressive Burkitt's tumors in that regressive tumors expressed larger levels of TNF-alpha, IL-6, IFN-gamma, IP-10, Mig, and IL-12 p35, but not other chemokines/cytokines. IP-10 and IL-12 were found to act as potent inhibitors of angiogenesis in vivo. IL-12 is an inducer of IFN-gamma and indirectly of IP-10, an IFN-gamma-inducible protein, raising the possibility that the antiangiogenic effect of IL-12 is mediated by IP-10. Previous studies have demonstrated that IL-12 has potent antitumor activity in vivo. Much of this activity was dependent on the presence of IFN-gamma and of immune T cells. The observation, made in our studies, that IP-10 is an inhibitor of angiogenesis raises the possibility that IP-10 might contribute to the antitumor effects of IL-12 by inhibiting angiogenesis. Inhibition of angiogenesis by IP-10 and IL-12 is T-cell independent, suggesting that IL-12 targets at least two compartments, T cells and capillaries, each capable of mediating antitumor effects.

Tyrosine phosphatase inhibition permits analysis of signal transduction complexes in p185HER2/neu-overexpressing human tumor cells.

The HER2/neu gene encodes a receptor tyrosine kinase that is highly homologous to the epidermal growth factor receptor. Overexpression of the receptor in mammary and ovarian carcinoma correlates with poor patient prognosis. To determine how the overexpression of a normal receptor leads to the generation of an oncogenic signal, we compared the patterns of tyrosine phosphorylation in tumor-derived human cell lines expressing high levels of p185HER2/neu. In intact SKBR3 cells, basal phosphorylation of p185HER2/neu was not detected. However, pretreatment of cells with the tyrosine phosphatase inhibitor, sodium orthovanadate, led to the detection of phosphotyrosine on phospholipase C-gamma (PLC-gamma), GTPase-activating protein but not on the RAF-1 kinase. Strikingly, PLC-gamma was detected in a complex which contained multiple tyrosine-phosphorylated polypeptides. This complex was detected only in cytoplasmic fractions and had a distinct composition in different p185HER2/neu-overexpressing cell lines. Although GTPase-activating protein has been found previously in association with proteins of 190 and 62 kDa in fibroblasts, in SKBR3 cells it was found associated with multiple additional tyrosine-phosphorylated polypeptides. These experiments show that SKBR3 cells possess high levels of protein tyrosine phosphatase that can act upon p185HER2/neu. Moreover, they reveal, for the first time, the presence of PLC-gamma and GTPase-activating protein in cytosolic complexes containing a variety of other tyrosine-phosphorylated polypeptides. These observations suggest novel possibilities for the specific definition of receptor-generated signals in tumor cells.

Effects of dibutyryl-cAMP on adenovirus replication in different cell lines

Replication of adenovirus type 2 (Ad2) was inhibited by dibutyryl-cAMP (db-cAMP) in three fibroblastic cell lines, BHK21, HEK, and WI-38, and in Ad12 transformed BHK21 cells with high intracellular level of cAMP. Yield of Ad12 in WI-38 cells was also reduced. The inhibitory effects were undiminished when db-cAMP was added up to 9 h.p.i. but decreased on later additions. In contrast, replication of either Ad2 or Ad12 was not affected by db-cAMP in two tumor-derived human cell lines, HeLa and KB.