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.

Data on the inhibition of RNase inhibitor activity by a monoclonal antibody as assessed by microfluidics-based RNA electrophoresis.

Using purified reaction components, a commercial monoclonal antibody (Ab) specific to RNase inhibitor (RI) was found to interfere with the activity of RI. Total RNA was mixed with a monoclonal Ab specific to either RI (clone 3F11) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNase A, RI, or a combination of the above. Following incubation for 1 h at 22 °C or 37 °C, RNA integrity of the mixtures was assessed using microfluidics-based Bio-Rad Experion RNA electrophoresis. The addition of Ab 3F11 prevented RI from effectively inhibiting RNase A and therefore resulted in extensive RNA degradation. The data presented are associated with the research article entitled "Endogenous RNase Inhibitor Contributes to Stability of RNA in Crude Cell Lysates: Applicability to Reverse Transcription Quantitative PCR (RT-qPCR)" (Wang et al., 2016) [1].

Endogenous RNase inhibitor contributes to stability of RNA in crude cell lysates: Applicability to RT-qPCR.

Crude cell lysates are increasingly used as input for direct analysis by reverse transcription quantitative PCR (RT-qPCR), particularly for high-throughput applications. We previously demonstrated that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercial cell-lysis reagents for the preparation of RT-qPCR-ready cell lysates; addition of an exogenous RNase inhibitor (RI) to the lysis buffer was found to be unnecessary to maintain RNA stability in cell lysates either freshly prepared or previously stored frozen at -80 °C. In the present study, we have demonstrated that the stability of RNA observed in our cell lysates is due to the presence of the endogenous RI. Furthermore, we have established the generalizability and applicability of this phenomenon by evaluating lysates prepared from cell lines commonly used in virology (A549, HeLa, MDCK, and Vero). Awareness of the mechanism underlying RNA stability may engender greater confidence in generating cell lysates for RT-qPCR without relying on addition of exogenous RI (a substantial cost-saving benefit) and encourage appropriate practices for handling and storage of samples.

MicroRNAs as potential biomarkers for VERO cell tumorigenicity.

MicroRNA 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.

A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR.

Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription qPCR (RT-qPCR). The need for cumbersome RNA purification is circumvented in our assays by making use of a commercial reagent that can easily generate crude cell lysates amenable to direct analysis by one-step RT-qPCR. In the present study, we demonstrate that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercially available reagents for the purpose of generating RT-qPCR-ready cell lysates from MDCK cells infected with influenza virus. We have found that addition of exogenous RNase inhibitor as a buffer component is not essential in order to maintain RNA integrity, even following stress at 37 °C incubation for 1-2 hours, in cell-lysate samples either freshly prepared or previously stored frozen at -80 °C.

Failure-to-thrive syndrome associated with tumor formation by Madin-Darby canine kidney cells in newborn nude mice.

Tumors that formed in newborn nude mice that were inoculated with 10(7) Madin-Darby canine kidney (MDCK) cells were associated with a failure-to-thrive (FTT) syndrome consisting of growth retardation, lethargy, weakness, and dehydration. Scoliosis developed in 41% of affected pups. Pups were symptomatic by week 2; severely affected pups became moribund and required euthanasia within 3 to 4 wk. Mice with FTT were classified into categories of mild, moderate, and severe disease by comparing their weight with that of age-matched normal nude mice. The MDCK-induced tumors were adenocarcinomas that invaded adjacent muscle, connective tissue, and bone; 6 of the 26 pups examined had lung metastases. The induction of FTT did not correlate with cell-line aggressiveness as estimated by histopathology or the efficiency of tumor formation (tumor-forming dose 50% endpoint range = 10(2.8) to 10(7.5)); however, tumor invasion of the paravertebral muscles likely contributed to the scoliosis noted. In contrast to the effect of MDCK cells, tumor formation observed in newborn mice inoculated with highly tumorigenic, human-tumor-derived cell lines was not associated with FTT development. We suggest that tumor formation and FTT are characteristics of these MDCK cell inocula and that FTT represents a new syndrome that may be similar to the cachexia that develops in humans with cancer or other diseases.

A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment.

BACKGROUND: Few studies have used quantitative polymerase chain reaction (qPCR) as an approach to measure virus neutralization assay endpoints. Its lack of use may not be surprising considering that sample nucleic acid extraction and purification can be expensive, labor-intensive, and rate-limiting. METHODS: Virus/antibody mixtures were incubated for one hour at 37°C and then transferred to Vero cell monolayers in a 96-well plate format. At 24 (or 48) hours post-infection, we used a commercially available reagent to prepare cell lysates amenable to direct analysis by one-step SYBR Green quantitative reverse transcription PCR using primers specific for the RSV-N gene, thereby obviating the need for cumbersome RNA extraction and purification. The neutralization titer was defined as the reciprocal of the highest dilution needed to inhibit the PCR signal by 90% when compared with the mean value observed in virus control wells in the absence of neutralizing antibodies. RESULTS: We have developed a qPCR-based neutralization assay for human respiratory syncytial virus. Due to the sensitivity of qPCR in detecting virus replication, endpoints may be assessed as early as 24 hours post-infection. In addition, the dynamic range of qPCR provides a basis for the assay to be relatively robust to perturbations in input virus dose (i.e., the assay is in compliance with the Percentage Law). CONCLUSIONS: This qPCR-based neutralization assay is suitable for automated high-throughput applications. In addition, our experimental approach may be generalizable for the rapid development of neutralization assays for other virus families.

Development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription PCR.

A microneutralization assay using an ELISA-based endpoint assessment (ELISA-MN) is widely used to measure the serological response to influenza virus infection and vaccination. We have developed an alternative microneutralization assay for influenza virus using a quantitative reverse transcription PCR-based endpoint assessment (qPCR-MN) in order to improve upon technical limitations associated with ELISA-MN. For qPCR-MN, infected MDCK-London cells in 96-well cell-culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR (qRT-PCR; SYBR Green chemistry with primers targeting a conserved region of the M1 gene of influenza A viruses). The growth curves of three recent vaccine strains demonstrated that the qRT-PCR signal detected at 6 hours post-infection reflected an amplification of at least 100-fold over input. Using ferret antisera, we have established the feasibility of measuring virus neutralization at 6 hours post-infection, a duration likely confined to a single virus-replication cycle. The neutralization titer for qPCR-MN was defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal; this endpoint was found to be in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN was robust with respect to assay duration (6 hours vs. 12 hours). In addition, qPCR-MN appeared to be compliant with the Percentage Law (i.e., virus neutralization results appear to be consistent over an input virus dose ranging from 500 to 12,000 TCID(50)). Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and inter-laboratory variability while affording suitability for automation and high-throughput uses. Finally, our qRT-PCR-based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses.

Heterogeneity of the tumorigenic phenotype expressed by Madin-Darby canine kidney cells.

The mechanisms by which cells spontaneously immortalized in tissue culture develop the capacity to form tumors in vivo likely embody fundamental processes in neoplastic development. The evolution of Madin-Darby canine kidney (MDCK) cells from presumptively normal kidney cells to immortalized cells that become tumorigenic represents an example of neoplastic development in vitro. Studies of the mechanisms by which spontaneously immortalized cells develop the capacity to form tumors would benefit from quantitative in vivo assays. Most mechanistic correlations are evaluated by using single-dose tumor-induction experiments, which indicate only whether cells are or are not tumorigenic. Here we used quantitative tumorigenicity assays to measure dose-and time-dependent tumor development in nude mice of 3 lots of unmodified MDCK cells. The results revealed lot-to-lot variations in the tumorigenicity of MDCK cells, which were reflected by their tumor-inducing efficiency (threshold cell dose represented by mean tumor-producing dose; log(10) 50% endpoints of 5.2 for vial 1 and 4.4 for vial 2, and a tumor-producing dose of 5.8 for vial 3) and mean tumor latency (vial 1,6.6 wk; vial 2,2.9 wk; and vial 3,3.8 wk). These studies provide a reference for further characterization of the MDCK cell neoplastic phenotype and may be useful in delineating aspects of neoplastic development in vitro that determine tumor-forming capacity. Such data also are useful when considering MDCK cells as a reagent for vaccine manufacture.

Patterns of microRNA expression in non-human primate cells correlate with neoplastic development in vitro.

MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression post-transcriptionally. They play a critical role in developmental and physiological processes and have been implicated in the pathogenesis of several diseases including cancer. To identify miRNA signatures associated with different stages of neoplastic development, we examined the expression profile of 776 primate miRNAs in VERO cells (a neoplastically transformed cell line being used for the manufacture of viral vaccines), progenitor primary African green monkey kidney (pAGMK) cells, and VERO cell derivatives: spontaneously immortalized, non-tumorigenic, low-passage VERO cells (10-87 LP); tumorigenic, high-passage VERO cells (10-87 HP); and a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice. When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells. We identified 10 up-regulated miRNAs whose level of expression correlated with VERO cell evolution from a non-tumorigenic phenotype to a tumorigenic phenotype. The overexpression of miR-376a and the polycistronic cluster of miR-376a, miR-376b and miR-376c conferred phenotypic changes to the non-tumorigenic 10-87 LP cells that mimic the tumorigenic 10-87 HP cells. Thirty percent of miRNAs that were components of the identified miRNAs in our spontaneously transformed AGMK cell model are also dysregulated in a variety of human tumors. These results may prove to be relevant to the biology of neoplastic development. In addition, one or more of these miRNAs could be biomarkers for the expression of a tumorigenic phenotype.

A quantitative PCR assay for SV40 neutralization adaptable for high-throughput applications.

A neutralization assay incorporating a quantitative SYBR Green PCR endpoint has been developed for SV40. The present study demonstrates that crude virus samples can serve as suitable amplification templates for quantitative PCR without the need for nucleic acid extraction. The denaturation temperature of thermocycling appears to be sufficient to release the encapsidated viral genome and allow its availability as a PCR template. Issues arising from inhibitors of PCR present in crude virus samples can be circumvented easily by a 100-fold dilution step. Using a streamlined procedure that eliminates sample nucleic acid extraction (a hitherto rate-limiting step that diminishes throughput substantially), quantitative PCR was applied in order to assess: (1) the replication kinetics of SV40 and (2) the inhibition of SV40 productive infection by neutralizing antibodies. A similar high-throughput approach might be feasible for related polyomaviruses (e.g., BKV and JCV) as well as for other families of viruses.

Identification of a neutralization epitope in the VP1 capsid protein of SV40.

Three SV40 escape mutants were identified by selection in the presence of monoclonal antibodies with neutralizing activity. The VP1 amino acid alterations in these mutants were: (1) K73-->E (in loop BC); (2) D77-->E (in loop BC); (3) K171-->R (in loop EF); and (4) Q175-->H (in loop EF). These residues are clustered in close proximity to each other on the surface of the native capsid protein, strongly suggesting that they form a conformational epitope directly recognized by the neutralizing antibody. To our knowledge, the present study represents the first experimental mapping of a neutralization epitope of a polyomavirus family member. Structural information regarding the neutralization epitope should be useful for clarifying the extent of cross-reactivity exhibited by the humoral immune response towards related primate polyomaviruses (e.g., SV40, BKV, and JCV).

Testing an ecological model for transmission of Salmonella enterica in swine production ecosystems using genotyping data.

An ecological model for transmission of Salmonella enterica in swine production ecosystems was developed, identifying host species, environmental reservoirs, and temporal, spatial, and functional (i.e., stage of production) dimensions. It was hypothesized that transmission was most likely within spatial and functional compartments, between hosts of the same species and abiotic compartments of the same type. Eighteen swine production systems in Illinois, USA, were sampled in four collection cycles (1998, 1999, 2000, 2003). There were 11,873 samples collected, including feces from swine and other mammals and birds, and samples from insects, pen floors, boots, feed, and water. The 460 Salmonella isolates obtained were genotyped using repetitive sequence PCR with three primers-REP, BOX, and ERIC. All isolates from 2000 and 2003 were serotyped, as well as a subsample from 1998 and 1998. Genetic relatedness was estimated from the similarity of fragmentation patterns after gel electrophoresis of PCR products. Cluster analysis identified genetically related isolates. Linking of isolates in tight clusters (similarity >or=85%) was viewed as evidence for transmission. Five farms had a sufficient number of tight clusters for hypothesis testing. The factors most differentiating isolates genetically were farm of origin and time of sampling. Isolates were also differentiated genetically by site, building, room, and pen. There was no consistent association of genotype with stage of production or host/environment reservoir. Serotype analysis confirmed that Salmonella lineages were differentiated by visit and site. Thus, Salmonella transmission was primarily over short distances, i.e., within the same pen or room, with some transmission between rooms and buildings on the same site, but with limited transmission between sites. Transmission was observed across a variety of ecological niches represented by different host species and environmental reservoirs. Genetic differences over time reflected multiple introductions into the ecosystem of different Salmonella genotypes, as well as evolutionary changes within lineages. Intervention strategies to reduce Salmonella prevalence within swine production ecosystems would be best targeted at maintaining spatial barriers to transmission, whereas intervention targeted at specific biological hosts or environmental reservoirs is less likely to be effective.

Mechanism of action of a distal NF-kappaB-dependent enhancer.

The monocyte chemoattractant protein 1 gene (MCP-1) is regulated by TNF through an NF-kappaB-dependent distal enhancer and an Sp1-dependent promoter-proximal regulatory region. In the silent state, only the distal regulatory region is accessible to transcription factors. Upon activation by tumor necrosis factor, NF-kappaB binds to the distal regulatory region and recruits CBP and p300. CBP and p300 recruitment led to specific histone modifications that ultimately enabled the binding of Sp1 to the proximal regulatory region. During this process, a direct interaction between the distal and proximal regulatory regions occurred. Sp1, NF-kappaB, CBP, and p300 were required for this interaction. CBP/p300-mediated histone modifications enhanced the binding of the coactivator CARM1 to the distal regulatory region. CARM1, which is necessary for MCP-1 expression, was not required for distal-proximal region interactions, suggesting that it plays a later downstream activation event. The results describe a model in which the separation of the distal enhancer from the promoter-proximal region allows for two independent chromatin states to exist, preventing inappropriate gene activation at the promoter while at the same time allowing rapid induction through the distal regulatory region.

Comparison of pulsed field gel electrophoresis and repetitive sequence polymerase chain reaction as genotyping methods for detection of genetic diversity and inferring transmission of Salmonella.

Pulsed field gel electrophoresis (PFGE) using restriction enzymes AvrII, SpeI, and XbaI, and repetitive sequence polymerase chain reaction (Rep-PCR) using BOX, ERIC, and REP primers, were compared with respect to their ability to detect genetic differences among 68 Salmonella isolates from nine Illinois swine farms. Both genotyping methods had high reproducibility of fragment numbers (reliability>0.9) and sizes (reliability>0.85), and sizes [Formula: see text], and produced approximately the same number of DNA fragments, but Rep-PCR fragment profiles had considerably greater variation. Genetic distances between isolates were calculated from fragment size matching. There was good agreement between the genetic distance matrices for the composite (3-enzyme and 3-primer) methods (Mantel's r=0.83). PFGE detected slightly greater variation in genetic distances among isolates, but failed to differentiate seven pairs of isolates, three of which were sampled at least 1 month apart and therefore unlikely to be truly identical genetically. In contrast, Rep-PCR identified no isolates as genetically identical. In cluster analyses based on genetic distances, there were moderate differences between PFGE and Rep-PCR (about 2/3 agreement in tight cluster membership). Both PFGE and Rep-PCR were able to differentiate isolates of the same serotype. However, some serotypes (Agona, Anatum, Derby, Infantis, Worthington) were distributed across clusters. There was less agreement between individual primer/enzyme and composite results for Rep-PCR than for PFGE. This greater independence of results for individual primers for Rep-PCR accounted in part for the greater discriminative ability of the composite method. Both composite methods indicated that most Salmonella transmission occurred within a farm and that there was no preference for transmission between specific ecological compartments. Given the equally high reliability of both genotyping methods, the greater discriminative ability of Rep-PCR recommends it as the preferred method for precise detection of transmission links.