Beta-actin in human colon adenocarcinoma cell lines with different metastatic potential

Human colon adenocarcinoma LS180 parental cell line and selected variants, characterized by different metastatic capacity were used to examine, whether a correlation exists between beta-actin expression, its subcellular distribution and metastatic potential of these cells. Cytosolic fraction (supernatant 105000 x g), isolated from the tumor cells was used as a source for actin quantification. The higher level of beta-actin was observed in the cytosol of three selected sublines to compare with LS180 parental line. Statistically significant increase of beta-actin level in highly motile EB3 cells variant should be underlined to compare with the other sublines. Distinct differences in the phenotype of adenocarcinoma cell variants were found, such as the changes in cells shape, cells spreading and ability to attach to the surface of culture dish. Actin cytoskeleton was visualized with fluorescence microscopy application and microfilaments rhodamine-conjugated phalloidin staining. beta-actin subcellular localization was done by immunofluorescence staining with monoclonal anti-beta actin antibodies. In the elongated cells (LS180, 3LNLN), this isoactin is dispersed in the whole cell body and concentrates in pseudopods and at the leading edges, when in the rounded variant (EB3) beta-actin dominates mainly in cortical ring under cellular membrane and it is also seen in the subtle protrusions. Summary of our former (Nowak et al., 2002, Acta Biochim. Polon., 49: 823) and current data lead to the conclusion that there is a distinct correlation between metastatic capacity of examined human colon adenocarcinoma cells, the state of actin polymerization, actin cytoskeleton organization and beta-actin expression.     

Splicing mutations in TP53 in human squamous cell carcinoma lines influence immunohistochemical detection.

The mutational status of the tumor suppressor gene TP53 is often examined by immunohistochemistry. We compared the incidence of TP53 mutations in 12 permanent squamous cell carcinoma lines of the head and neck with the immunohistochemical staining obtained with two different antibodies. The mutational status of the TP53 gene was assessed by sequencing the complete coding frame of the TP53 mRNA. All 12 tumor cell lines had TP53 mutations. Six of them showed missense mutations and five had premature stop codons caused either by splicing mutations or nonsense mutations or by exon skipping. One tumor cell line was heterozygous, with a truncating splicing mutation and an additional missense mutation located on different alleles. In one case, an in-frame insertion of 23 extra codons was found. All missense mutations were positive in immunhistochemistry and Western blotting. The truncated p53 was not immunohistochemically detected in three cases with the DO-7 antibody and in five cases with the G59-12 antibody, giving false-negative results in 25% or 40%, respectively, of all tumor cell lines examined. We conclude that splicing mutations are common in squamous cell carcinoma lines and that the incidence of p53 inactiviation by erroneous splicing is higher than yet reported. Sequencing of only the exons of TP53 may miss intronic mutations leading to missplicing and may therefore systematically underestimate the TP53 mutation frequency.     

High levels of ezrin expressed by human pancreatic adenocarcinoma cell lines with high metastatic potential.

Ezrin is a membrane cytoskeleton crosslinker protein that is a member of the ERM (ezrin/radixin/moesin) family. Ezrin binds adhesion molecules such as CD43, CD44, ICAM-1, and ICAM-2, which are implicated in cell migration and metastasis. Ezrin is expressed by many tumor cell lines; however, little is known about the function of ezrin in tumorigenesis and metastasis. Here, we investigated expression of ezrin in pancreatic adenocarcinoma cell lines of different metastatic potential. Among 16 pancreatic adenocarcinoma cell lines, several cell lines showed strong expression of ezrin. Two cell lines with high metastatic potential, S2-CP9 and S2-VP10, showed very high levels of ezrin mRNA and protein, whereas other sublines showed lower levels. There was no relationship between the expression levels of ezrin and the differentiation grades of the cell lines. These results suggest that there is a relationship between high expression of ezrin and metastatic potential of pancreatic carcinomas.     

Transglutaminase activity and putrescine-binding capacity in cloned cell lines with different metastatic potential

An inverse correlation was found between cellular transglutaminase activity and metastatic potential of four cloned cell lines derived from a primary nickel-induced rat rhabdomyosarcoma. Cellular transglutaminase activity as assessed with endogenous cellular protein or exogenous methylated casein was greatest in the clone F9-4/8 which is the least metastasizing. When the putrescine-binding capacity of one cellular derived protein - fibronectin - was examined with exogenous transglutaminase, it was found that the fibronectin derived from the clone F9-4/8 showed the lowest binding capacity compared with those from the other clones. However, when the overall binding capacity of cellular proteins from each cell line was examined no differences could be detected. The results are discussed in the light of the well-known role of fibronectin in cellular adhesion.     

Intracellular zinc increase inhibits p53(-/-) pancreatic adenocarcinoma cell growth by ROS/AIF-mediated apoptosis

We show that treatment with non-toxic doses of zinc in association to the ionophore compound pyrrolidine dithiocarbamate (PDTC) inhibits p53(-/-) pancreatic cancer cell growth much more efficiently than gemcitabine, the gold standard chemotherapeutic agent for pancreatic cancer. Both the metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine and the radical scavenger N-acetyl-l-cysteine are able to recover cell growth inhibition by Zn/PDTC, demonstrating that this effect depends on the increased levels of intracellular zinc and of reactive oxygen species (ROS). Zn/PDTC treatment induces a strong apoptotic cell death that is associated to ROS-dependent nuclear translocation of the mitochondrial factor AIF, but not to the regulation of apoptotic genes and caspase activation. Primary fibroblasts are more resistant than pancreatic cancer cells to Zn/PDTC treatment and exhibit a lower basal and Zn/PDTC-induced enhancement of intracellular zinc. We show that Zn/PDTC induces p53 proteasomal degradation and that the proteasome inhibitor MG132 further increases fibroblast growth inhibition by Zn/PDTC, suggesting that p53 degradation plays an important role in fibroblast resistance to Zn/PDTC.     

Establishment of mouse embryonic stem cell-derived erythroid progenitor cell lines able to produce functional red blood cells

Background

The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If erythroid cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. To evaluate the feasibility of establishing useful erythroid cell lines, we attempted to establish such cell lines from mouse embryonic stem (ES) cells.

Methodology/Principal Findings

We developed a robust method to obtain differentiated cell lines following the induction of hematopoietic differentiation of mouse ES cells and established five independent hematopoietic cell lines using the method. Three of these lines exhibited characteristics of erythroid cells. Although their precise characteristics varied, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. In addition, we did not observe formation of any tumors following transplantation of these cells.

Conclusion/Significance

To the best of our knowledge, this is the first report to show the feasibility of establishing erythroid cell lines able to produce mature RBCs. Considering the number of human ES cell lines that have been established so far, the intensive testing of a number of these lines for erythroid potential may allow the establishment of human erythroid cell lines similar to the mouse erythroid cell lines described here. In addition, our results strongly suggest the possibility of establishing useful cell lines committed to specific lineages other than hematopoietic progenitors from human ES cells.     

Induction of accelerated senescence by gamma radiation in human solid tumor-derived cell lines expressing wild-type TP53

Recent studies have demonstrated that p21WAF1 (now known as CDKN1A)-dependent and -independent accelerated senescence responses are a major determinant of the sensitivity of cancer cells to chemotherapeutic agents. The objective of the present study was to determine whether human solid tumor-derived cell lines that express wild-type TP53 can exhibit levels of CDKN1A induction after exposure to ionizing radiation that are sufficient to activate the accelerated senescence program. Exposure to 60Co gamma radiation (< or =8 Gy) triggered accelerated senescence in all five TP53 wild-type tumor cell lines examined, albeit to differing degrees. Three of the TP53 wild-type tumor cell lines, HCT116, A172 and SKNSH, activated the TP53 signaling pathway similarly to normal human fibroblasts, as judged by the nuclear accumulation of TP53, magnitude and duration of induction of CDKN1A mRNA and CDKN1A protein, and propensity to undergo accelerated senescence after radiation exposure. In the clonogenic survival assay, the degree of radiosensitivity of these three tumor cell lines was also in the range displayed by normal human fibroblasts. On the other hand, two other TP53 wild-type tumor cell lines, A498 and A375, did not maintain high levels of CDKN1A mRNA and CDKN1A protein at late times postirradiation and exhibited only low levels of accelerated senescence after radiation exposure. Studies with a CDKN1A knockout cell line (HCT116CDKN1A-/-) confirmed that the radiation-triggered accelerated senescence is dependent on CDKN1A function. We conclude that (1) clinically achievable doses of ionizing radiation can trigger CDKN1A-dependent accelerated senescence in some human tumor cell lines that express wild-type TP53; and (2) as previously documented for normal human fibroblasts, some TP53 wild-type tumor cell lines (e.g. HCT116, A172 and SKNSH) may lose their clonogenic potential in response to radiation-inflicted injury primarily through undergoing accelerated senescence.     

Subcellular distribution and expression of cofilin and ezrin in human colon adenocarcinoma cell lines with different metastatic potential

The dynamic reorganization of the actin cytoskeleton is regulated by a number of actin binding proteins (ABPs). Four human colon adenocarcinoma cell lines – parental and three selected sublines, which differ in motility and metastatic potential, were used to investigate the expression level and subcellular localization of selected ABPs. Our interest was focused on cofilin and ezrin. These proteins are essential for cell migration and adhesion. The data received for the three more motile adenocarcinoma sublines (EB3, 3LNLN, 5W) were compared with those obtained for the parental LS180 adenocarcinoma cells and fibroblastic NRK cells. Quantitative densitometric analysis and confocal fluorescence microscopy were used to examine the expression levels and subcellular distribution of the selected ABPs. Our data show distinct increase in the level of cofilin in adenocarcinoma cells accompanied by the reduction of inactive phosphorylated form of cofilin. In more motile cells, cofilin was accumulated at cellular periphery in co-localization with actin filaments. Furthemore, we indicated translocation of ezrin towards the cell periphery within more motile cells in comparison with NRK and parental adenocarcinoma cells.In summary, our data indicate the correlation between migration ability of selected human colon adenocarcinoma sublines and subcellular distribution as well as the level of cofilin and ezrin. Therefore these proteins might be essential for the higher migratory activity of invasive tumor cells.Key words: actin, cofilin, ezrin, colon adenocarcinoma.     

7-Hydroxystaurosporine (UCN-01) preferentially sensitizes cells with a disrupted TP53 to gamma radiation in lung cancer cell lines

Mutations in TP53 occur in more than 50% of the lung cancer patients and are associated with an increased resistance to chemotherapy and radiotherapy. The human lung adenocarcinoma cell lines A549 and LXSN contain a wild-type TP53 and were growth arrested at both the G(1)- and G(2)-phase checkpoints after irradiation. However, a TP53-disrupted cell line, E6, was arrested only at the G(2)-phase checkpoint. UCN-01 (7-hydroxystaurosporine), a CHEK1 inhibitor that abrogates the G(2) block, has been reported to enhance radiation toxicity in human lymphoma and colon cancer cell lines. In this study, UCN-01 preferentially enhanced the radiosensitivity of the TP53-disrupted E6 cells compared to the TP53 wild-type cells. This effect was more pronounced in cells synchronized in early G(1) phase, where the E6 cells showed a higher resistance to radiation in the absence of drug. These results indicate that the combination of UCN-01 and radiation can more specifically target resistant TP53 mutated cancer cells and spare TP53 wild-type normal cells.     

Use of the comet-FISH assay to demonstrate repair of the TP53 gene region in two human bladder carcinoma cell lines

The alkaline single-cell gel electrophoresis (comet) assay can be combined with fluorescence in situ hybridization (FISH) methodology to investigate the localization of specific gene domains within an individual cell. The position of the fluorescent hybridization spots in the comet head or tail indicates whether the sequence of interest lies within or in the vicinity of a damaged region of DNA. In this study, we used the comet-FISH assay to examine initial DNA damage and subsequent repair in the TP53 gene region of RT4 and RT112 bladder carcinoma cells after 5 Gy gamma irradiation. In addition to standard comet parameter measurements, the number and location of TP53 hybridization spots within each comet was recorded at each repair time. The results indicate that the rate of repair of the TP53 gene region was fastest during the first 15 min after damage in both cell lines. When compared to overall genomic repair, the repair of the TP53 gene region was observed to be significantly faster during the first 15 min and thereafter followed a rate similar to that for the overall genome. The data indicate that the TP53 domain in RT4 and RT112 cells is repaired rapidly after gamma irradiation. Furthermore, this repair may be preferential compared to the repair of overall genomic DNA, which gives a measure of the average DNA repair response of the whole genome. We suggest that the comet-FISH assay has considerable potential in the study of gene-specific repair after DNA damage.     

Transfection of metastatic capability with total genomic DNA from human and mouse metastatic tumour cell lines

Abstract. From a large series of experiments involving transfer of high molecular weight total genomic DNA from highly metastatic human and mouse tumour cell lines to other mouse tumour cell lines we have derived a few cell lines with greatly augmented metastatic properties. In one of these experiments the transfected cell line (designated AH8 Test) not only colonised the lungs but also formed secondary tumour colonies in several extrapulmonary sites including the skin, skeletal muscles, bone, liver diaphragm, spleen and heart, There were no qualitative and quantitative effects of this magnitude when we used DNA from several non-metastatic or non-tumourigenic sources.
Secondary transfection of metastatic capability with DNA obtained from a metastasis formed by one of the primary transfectant lines (AH8 Test) has also been accomplished. Concomitant transfer of human DNA through both transfection cycles in this experiment was confirmed by a variety of methods including Southern blot analysis, in situ hybridisation and polymerase chain reaction (PCR) amplification of DNA using primers recognising human-specific Alu repeat sequences.
The findings offer opportunities for the isolation of sequences programming metastatic behaviour and we have cloned and sequenced a fragment of human DNA, which has not been previously characterised, from the transfected cells.     

Prognostic significance of TP53 mutations in oral squamous cell carcinoma with human papilloma virus infection

PURPOSE: The aim of this study was to analyze the prognostic impact of mutated TP53 in patients with oral squamous cell carcinoma (OSCC) whose tumors were infected with human papillomavirus (HPV). METHODS: Thirty-two HPV-positive OSCC patients were included. Most of them were clinically classified as stage III (n=29). All patients underwent postoperative radiotherapy (follow-up from 12 to 60 months, median 32). There were 21 relapses. DNA was isolated by phenol extraction from tumor tissue. HPV DNA (type 16, 18, 31, 33) was detected in genomic DNA of the tumors by the PCR-PAGE method. TP53 mutations (exons 4-8) were detected by the PCR-SSCP method. RESULTS: A statistically significant difference in the number of relapses in HPV-infected (13/21) versus HPV-infected and TP53-mutated (8/8) patients was observed. Patients with both TP53 mutation and HPV infection had a significantly shorter disease-free interval than patients with HPV infection only (median 6 versus 31 months, respectively). CONCLUSIONS: TP53 mutations are associated with a higher risk of relapse and contribute to an even worse prognosis of patients with OSCC when the tumors are HPV infected. The shorter disease-free interval in patients with TP53 mutations indicates that the response to postoperative radiotherapy may be influenced by TP53 status. The presence of both HPV infection and TP53 mutations may define a particular group of tumors with a more aggressive phenotype in advanced OSCC.     

Human TP53 from the malignant glioma-derived cell lines M059J and M059K has a cancer-associated mutation in exon 8

The protein coding segment of the TP53 genes from the glioma-derived cell lines M059J and M059K was sequenced. The sequences from both cell lines were identical over 5039 bp, including the gene segment containing exons 2 through 9, exon 10, and the proximal segment of exon 11. In both cells, the first nucleotide of codon 286 (GAA, Glu) is changed to an A (AAA, Lys). Comparison with the same TP53 segment from the A549 human lung carcinoma cell line revealed several differences in intron sequence.     

Screening for microorganism with cell wall lytic activity to produce protoplast-forming enzymes

Several different bacteria and fungi capable of degrading yeast cell walls were isolated in the course of a screening programme. One Streptomyces and one Acremonium strain were found to degrade yeast cell walls extremely well. Both isolates produced enzymes in liquid culture that could be used for protoplasting of Sporobolomyces salmonicolor (DSM 70851) and Rhodotorula rubra (DSM 70403). This fact is quite remarkable as, so far, S. salmonicolor could not be protoplasted by commercially available enzymes. Correspondence to: W. Kaul     

Establishment and characterization of human osteosarcoma cell lines with different pulmonary metastatic potentials

To develop an investigative tool for the study of human osteosarcoma (OSA), we established a human OSA cell line, namely, SOSP-9607, which exhibits a potential for spontaneous pulmonary metastasis. Subsequently, we screened two related sublines (F5M2 and F4) that have different pulmonary metastatic potentials. An in vivo orthotopic transplantation assay confirmed spontaneous pulmonary metastasis in all mice (100%) transplanted with the more aggressive OSA cells (F5M2) and a lesser degree of metastases with smaller nodules in 33.3% mice transplanted with the less aggressive OSA cell subline (F4). In mice transplanted with F5M2 cells, death from metastasis occurred at a median of 71 days; however, in mice transplanted with F4, no death occurred even after 120 days. Therefore, the F5M2 and F4 sublines, which originated from the same parent cell line, differed with respect to metastasis-related properties such as proliferating ability and invasiveness. Hence, these well-characterized human OSA sublines can be used as valuable models for comparative studies of genetic determinants of OSA in the future.     

Notch1 activation reduces proliferation in the multipotent hematopoietic progenitor cell line FDCP-mix through a p53-dependent pathway but Notch1 effects on myeloid and erythroid differentiation are independent of p53

Signaling mediated by activation of the transmembrane receptor Notch influences cell-fate decisions, differentiation, proliferation, and cell survival. Activated Notch reduces proliferation by altering cell-cycle kinetics and promotes differentiation in hematopoietic progenitor cells. Here, we investigated if the G(1) arrest and differentiation induced by activated mNotch1 are dependent on tumor suppressor p53, a critical mediator of cellular growth arrest. Multipotent wild-type p53-expressing (p53(wt)) and p53-deficient (p53(null)) hematopoietic progenitor cell lines (FDCP-mix) carrying an inducible mNotch1 system were used to investigate the effects of proliferation and differentiation upon mNotch1 signaling. While activated Notch reduced proliferation of p53(wt)-cells, no change was observed in p53(null)-cells. Activated Notch upregulated the p53 target p21(cip/waf) in p53(wt)-cells, but not in p53(null)-cells. Induction of the p21(cip/waf) gene by activated Notch was mediated by increased binding of p53 to p53-binding sites in the p21(cip/waf) promoter and was independent of the canonical RBP-J binding site. Re-expression of p53(wt) in p53(null) cells restored the inhibition of proliferation by activated Notch. Thus, activated Notch inhibits proliferation of multipotent hematopoietic progenitor cells via a p53-dependent pathway. In contrast, myeloid and erythroid differentiation was similarly induced in p53(wt) and p53(null) cells. These data suggest that Notch signaling triggers two distinct pathways, a p53-dependent one leading to a block in proliferation and a p53-independent one promoting differentiation.     

Establishment of adipose-derived mesenchymal stem cell lines from a p53-knockout mouse

Mesenchymal stem cells (MSCs) can differentiate into a variety of cell types. MSCs exist in several tissues such as the bone marrow, adipose, muscle, cartilage, and tendon. This differentiation potential makes MSCs candidates for cell-based therapeutic strategies for mesenchymal tissue injuries. MSCs can be prepared from bone marrow (BM-MSCs) and adipose (AD-MSCs); however, these MSCs exhibit senescence-associated growth arrest and display inevitable heterogeneity. We established several AD-MSC cell lines from a p53-knockout (KO) mouse. These cell lines were immortalized, but no cell lines grew anchorage-independently, suggesting that they are not cancerous. They differentiated into adipocytes, osteoblasts, and chondrocytes by treatment with certain stimuli. Moreover, following injection into the tail vein, the cells migrated into the wounded region of the liver and differentiated into hepatocytes. We succeeded in establishing several AD-MSC clonal cell lines that maintain the tissue-specific markers and characteristics of the developmental phase. These clonal cell lines will serve as important tools to study the mechanism of differentiation of MSCs.