MicroRNAs as potential biomarkers for VERO cell tumorigenicity

AbstractMicroRNA expression appears to capture the process of neoplastic development in vitro in the VERO line of African green monkey kidney (AGMK) cells (Teferedegne et al. PLoS One 2010;5(12):e14416). In that study, specific miRNA signatures were correlated with the transition, during serial tissue-culture passage, of low-density passaged 10–87 VERO cells from a non-tumorigenic phenotype at passage (p) 148 to a tumorigenic phenotype at p256. In the present study, six miRNAs (miR-376a, miR-654-3p, miR-543, miR-299-3p, miR-134 and miR-369-3p) were chosen from the identified signature miRNAs for evaluation of their use as potential biomarkers to track the progression of neoplastic development in VERO cells. Cells from the 10–87 VERO cell line at passage levels from p148 to p256 were inoculated into newborn and adult athymic nude mice. No tumors were observed in animals inoculated with cells from p148 to p186. In contrast, tumor incidences of 20% developed only in newborn mice that received 10–87 VERO cells at p194, p234 and p256. By qPCR profiling of the signature miRNAs of 10–87 VERO cells from these cell banks, we identified p194 as the level at which signature miRNAs elevated concurrently with the acquisition of tumorigenic phenotype with similar levels expressed beyond this passage. In wound-healing assays at 10-passage intervals between p150 to p250, the cells displayed a progressive increase in migration from p165 to p186; beginning at p194 and higher passages thereafter, the cells exhibited the highest rates of migration. By qPCR analysis, the same signature miRNAs were overexpressed with concomitant acquisition of the tumorigenic phenotype in another lineage of 10–87 VERO cells passaged independently at high density. Correlation between the passages at which the cells expressed a tumorigenic phenotype and the passages representing peaks in expression levels of signature miRNAs indicates that these miRNAs are potential biomarkers for the expression of the VERO cell tumorigenic phenotype

GLI1+ perivascular, renal, progenitor cells: The likely source of spontaneous neoplasia that created the AGMK1-9T7 cell line.

The AGMK1-9T7 cell line has been used to study neoplasia in tissue culture. By passage in cell culture, these cells evolved to become tumorigenic and metastatic in immunodeficient mice at passage 40. Of the 20 x 106 kidney cells originally plated, less than 2% formed the colonies that evolved to create this cell line. These cells could be the progeny of some type of kidney progenitor cells. To characterize these cells, we documented their renal lineage by their expression of PAX-2 and MIOX, detected by indirect immunofluorescence. These cells assessed by flow-cytometry expressed high levels of CD44, CD73, CD105, Sca-1, and GLI1 across all passages tested; these markers have been reported to be expressed by renal progenitor cells. The expression of GLI1 was confirmed by immunofluorescence and western blot analysis. Cells from passages 13 to 23 possessed the ability to differentiate into adipocytes, osteoblasts, and chondrocytes; after passage 23, their ability to form these cell types was lost. These data indicate that the cells that formed the AGMK1-9T7 cell line were GLI1+ perivascular, kidney, progenitor cells

A mouse strain defective in both T cells and NK cells has enhanced sensitivity to tumor induction by plasmid DNA expressing both activated H-Ras and c-Myc.

As part of safety studies to evaluate the risk of residual cellular DNA in vaccines manufactured in tumorigenic cells, we have been developing in vivo assays to detect and quantify the oncogenic activity of DNA. We generated a plasmid expressing both an activated human H-ras gene and murine c-myc gene and showed that 1 µg of this plasmid, pMSV-T24-H-ras/MSV-c-myc, was capable of inducing tumors in newborn NIH Swiss mice. However, to be able to detect the oncogenicity of dominant activated oncogenes in cellular DNA, a more sensitive system was needed. In this paper, we demonstrate that the newborn CD3 epsilon transgenic mouse, which is defective in both T-cell and NK-cell functions, can detect the oncogenic activity of 25 ng of the circular form of pMSV-T24-H-ras/MSV-c-myc. When this plasmid was inoculated as linear DNA, amounts of DNA as low as 800 pg were capable of inducing tumors. Animals were found that had multiple tumors, and these tumors were independent and likely clonal. These results demonstrate that the newborn CD3 epsilon mouse is highly sensitive for the detection of oncogenic activity of DNA. To determine whether it can detect the oncogenic activity of cellular DNA derived from four human tumor-cell lines (HeLa, A549, HT-1080, and CEM), DNA (100 µg) was inoculated into newborn CD3 epsilon mice both in the presence of 1 µg of linear pMSV-T24-H-ras/MSV-c-myc as positive control and in its absence. While tumors were induced in 100% of mice with the positive-control plasmid, no tumors were induced in mice receiving any of the tumor DNAs alone. These results demonstrate that detection of oncogenes in cellular DNA derived from four human tumor-derived cell lines in this mouse system was not possible; the results also show the importance of including a positive-control plasmid to detect inhibitory effects of the cellular DNA

Tumors Induced in Mice by Direct Inoculation of Plasmid DNA Expressing Both Activated H-ras and c-myc

<p>Vaccines contain residual DNA derived from the cells used to produce them. As part of our investigation to assess the risk of this cellular DNA, we are developing a quantitative in vivo assay to assess the oncogenicity of DNA. In an earlier study, we had generated expression plasmids for two oncogenes - human activated T24-H-<i>ras</i> and murine c-<i>myc</i> - and had shown that these two plasmids, pMSV-T24-H-<i>ras</i> and pMSV-c-<i>myc</i>, could act in concert to induce tumors in mice, although the efficiency was low. In this study, we took two approaches to increase the oncogenic efficiency: 1) both oncogene-expression cassettes were placed on the same plasmid; 2) transfection facilitators, which increase DNA uptake and expression <i>in vitro, </i>were tested. The dual-expression plasmid, pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i>, is about 20-fold more efficient at tumor induction in newborn NIH Swiss mice than the separate expression plasmids, with tumors being induced with 1 &#181;g of the dual-expression plasmid DNA. However, none of the transfection facilitators tested increased the efficiency of tumor induction. Based on these data, the dual-expression plasmid pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i> will be used as the positive control to develop a sensitive and quantitative animal assay that can be used to assess the oncogenic activity of DNA.</p

Evaluating the oncogenicity of DNA from cell lines established from four human tumors.

<p>Newborn CD3 epsilon mice were inoculated with 100 µg of cellular DNA from HeLa cells, CEM cells, A549 cells, or HT-1080 cells in the absence or presence of 1 µg of linear pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i> DNA. Tumors were found in 14/14 mice inoculated with HeLa DNA plus <i>ras</i>/<i>myc</i> DNA but in 0/55 inoculated with HeLa DNA alone; tumors were found in 19/19 mice inoculated with CEM DNA plus <i>ras</i>/<i>myc</i> DNA but in 0/55 inoculated with CEM DNA alone; tumors were found in 13/13 mice inoculated with A549 DNA plus <i>ras</i>/<i>myc</i> DNA but in 0/53 inoculated with A549 DNA alone; and tumors were found in 5/5 mice inoculated with HT-1080 DNA plus <i>ras</i>/<i>myc</i> DNA but in 0/19 inoculated with HT-1080 DNA alone. Tumors appeared within 8 weeks, and all were at the site of inoculation.</p

Histopathology of tumors induced by the <i>ras</i>/<i>myc</i> plasmid.

<p>Tumors induced by circular pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i> (panels <b>A</b> and <b>B</b>), and tumors induced by linear pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i> (panels <b>C</b>, <b>D</b>, <b>E</b>, and <b>F</b>). Tumors in panels <b>A</b> to <b>E</b> were undifferentiated sarcomas, while the tumor shown in panel F had the appearance of a differentiated fibrosarcoma.</p

Multiple tumors induced in newborn CD3 epsilon mice.

<p><b>A</b>. Three tumors evident in a newborn CD3 epsilon mouse that was inoculated SC with 25 µg of circular pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i> DNA. <b>B</b>. Seven tumors evident in a CD3 epsilon mouse inoculated SC with 1 µg of linear pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i> DNA; some of the tumors were not apparent until necropsy.</p