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.

The AGMK1-9T7 cell model of neoplasia: Evolution of DNA copy-number aberrations and miRNA expression during transition from normal to metastatic cancer cells.

To study neoplasia in tissue culture, cell lines representing the evolution of normal cells to tumor cells are needed. To produce such cells, we developed the AGMK1-9T7 cell line, established cell banks at 10-passage intervals, and characterized their biological properties. Here we examine the evolution of chromosomal DNA copy-number aberrations and miRNA expression in this cell line from passage 1 to the acquisition of a tumorigenic phenotype at passage 40. We demonstrated the use of a human microarray platform for DNA copy-number profiling of AGMK1-9T7 cells using knowledge of synteny to 'recode' data from human chromosome coordinates to those of the African green monkey. This approach revealed the accumulation of DNA copy-number gains and losses in AGMK1-9T7 cells from passage 3 to passage 40, which spans the period in which neoplastic transformation occurred. These alterations occurred in the sequences of genes regulating DNA copy-number imbalance of several genes that regulate endothelial cell angiogenesis, survival, migration, and proliferation. Regarding miRNA expression, 195 miRNAs were up- or down-regulated at passage 1 at levels that appear to be biologically relevant (i.e., log2 fold change >2.0 (q<0.05)). At passage 10, the number of up/down-regulated miRNAs fell to 63; this number increased to 93 at passage 40. Principal-component analysis grouped these miRNAs into 3 clusters; miRNAs in sub-clusters of these groups could be correlated with initiation, promotion, and progression, stages that have been described for neoplastic development. Thirty-four of the AGMK1-9T7 miRNAs have been associated with these stages in human cancer. Based on these data, we propose that the evolution of AGMK1-9T7 cells represents a detailed model of neoplasia in vitro.

Assessment of potential miRNA biomarkers of VERO-cell tumorigenicity in a new line (AGMK1-9T7) of African green monkey kidney cells.

Patterns of microRNA expression appear to delineate the process of spontaneous neoplastic development-transformation (SPNDT) occurring in the African green monkey kidney (AGMK) VERO cell line (Teferedegne et al., 2010). Analysis of microarray data identified 6 microRNAs whose high-level of expression peaked when the World Health Organization 10-87 VERO cells became tumorigenic at passage (p) 190. Six miRNAs were identified as potential biomarkers for the expression of the VERO-cell tumorigenic phenotype (Teferedegne et al., 2014). However, the question remained whether these miRNA biomarkers are specific for VERO cells or can be generalizable to other cells originating from African green monkey kidneys. To examine miRNA expression patterns in AGMK cells at lower passage levels and to re-examine the identified miRNAs as biomarkers associated with tumorigenic phenotype of VERO cells in another independently-derived line, we established a new line of African green monkey kidney cells (AGMK1-9T7) by serially passaging kidney cells from another AGM. The AGMK1-9T7 cells became tumorigenic in nude mice at p40. Evaluation of miRNA expression at intervals from p1 to p40 revealed similarities between the evolution of miRNA expression during SPNDT in the AGMK1-9T7 cells and the 10-87 VERO cells. Four of the 6 potential biomarker miRNAs (miR-376a, miR-654-3p, miR-543, miR-134) in our earlier reports were detected by microarray in the AGMK1-9T7 cells; RT-qPCR analysis detected all 6 miRNAs. All 6 of these miRNAs have been associated with human tumors. Detection of the same miRNAs associated with the tumorigenic p40 AGMK1-9T7 cells and tumorigenic 10-87 VERO cells confirmed our proposal that these miRNA represent biomarkers for the tumor-forming ability of AGMK/VERO cells. The similarities of expression of miRNAs in different AGMK cell lines that were established 50years apart suggest that the process of SPNDT in these non-human primate cells in tissue culture is based upon similar genetic and epigenetic mechanisms.