Assessing the tumorigenic phenotype of VERO cells in adult and newborn nude mice.

VERO cell lines are important substrates for viral vaccine manufacture. The mechanism by which these cells became neoplastically transformed is unknown. During tissue-culture passage, VERO cells can develop the capacity to form tumors. Although at the passage levels (around p140) currently used for vaccine manufacture, VERO cells are non-tumorigenic, questions have been raised about safety issues that might be associated with this capacity to acquire a tumorigenic phenotype. To begin to address these issues, the tumorigenicity of VERO cell lines, derived at different passage levels under different growth conditions, were evaluated in 365-day assays in adult and newborn nude mice. High passage (p>200) VERO cell lines established by random passaging in tissue culture produced tumors in adult (10 out of 27) mice and newborn (21 out of 30) mice, respectively. In contrast, a high passage (p>250) cell line established by passage at sub-confluence produced tumors only in newborn mice (16 out of 30). Progressively growing tumors began forming at 36 days in newborns and at 69 days in adults. Higher tumor incidences and shorter tumor latencies suggest that newborn nude mice may be more sensitive than adults in detecting the expression of a tumorigenic phenotype by some VERO cell lines.     

Differential expression of microRNAs in myometrium and leiomyomas and regulation by ovarian steroids

Given the emerging roles of microRNAs (miRNAs) as key regulator of mRNA stability we assessed their expression profile in paired myometrium and leiomyoma, their isolated smooth muscle cells (MSMC and LSMC), a spontaneously transformed leiomyoma smooth muscle cells (T-LSMC) and SK-LMS-1, a leiomyosarcoma cell line using microarray and real time PCR.Based on global normalization of expression values of 385 miRNAs and statistical analysis (anova), 91 miRNAs were expressed above the threshold levels in myometrium, with a progressive decline in numbers in leiomyomas, MSMC, LSMC, T-LSMC and SK-LMS-1 (P<0.05).We selected and validated the expression of miR-20a, miR-21, miR-26a, miR-18a, miR-206, miR-181a and miR-142-5p and found their differential expression in tissue and cell-specific manners (P<0.05).Treatments of MSMC and LSMC with 17beta estradiol and medroxyprogesterone acetate (10(-8)M), or ICI-182780 and RU-486 (10(-6)M) resulted in differential regulation of these miRNAs (P<0.05).In conclusion, the expression of a number of miRNAs in myometrium and leiomyoma with their progressive aberrant from normal MSMC into LSMC, transformed and cancerous stage, suggests that miRNAs and their regulation by ovarian steroids play a key role in pathogenesis of leiomyoma through gene expression stability.     

Correlation of tumorigenicity with resistance to growth inhibition by cis-hydroxyproline

cis-Hydroxyproline, an inhibitor of collagen deposition, was examined for its effect on the growth of a variety of tumorigenic and non-tumorigenic cells. Virally, chemically, and spontaneously transformed murine cell lines were found to be less sensitive to cell spreading and growth inhibition by cis-hydroxyproline (CHP) than were their non-tumorigenic counterparts. The non-tumorigenic lines exhibited a higher rate of collagen accumulation in culture than the tumorigenic cell lines. The rate of collagen accumulation in culture without CHP and the growth inhibition by CHP were directly related. These results suggest that normal but not tumorigenic cells may require synthesis of an extracellular substrate containing collagen to support spreading and growth.     

Conversion of premalignant human cells to tumorigenic cells by methylmethane sulfonate and methylnitronitrosoguanidine

Nine human tumor cell lines derived from both epithelial and mesenchymal tumors exhibited either an anchorage-independent growth non-tumorigenic phenotype or an anchorage-independent tumorigenic phenotype. Transformed epithelial cell lines with the non-tumorigenic phenotype could be converted to a progressively growing tumor phenotype following treatment with either methylmethane sulfonate (MMS) or N-methyl-N-nitro-N-nitrosoguanidine (MNNG). In contrast, sarcoma derived cell lines with a non-tumorigenic phenotype could be converted to a progressively growing tumor phenotype only with MNNG. SV40 immortalized HET-1A non-tumorigenic phenotype cells could be converted to a progressively growing tumorigenic phenotype, infrequently, when treated with MNNG, but not MMS. Progressively growing tumors produced by either MMS or MNNG treated non-tumorigenic phenotypes exhibited metastatic potential in nude mice. Chemically treated HET-1A cells acquired the ability to produce tumor in mice but the tumor did not exhibit metastatic potential. In contrast, populations of tumorigenic cells were not rendered more biologically aggressive after treatment with either MMS or MNNG; i.e., the latency period for tumor development was not accelerated and the tumors did not exhibit metastatic potential. These results suggest that the biological effects of MMS and MNNG on non-tumorigenic, tumorigenic and immortalized cell lines are phenotype specific.Abbreviations AIG anchorage-independent growth - DMSO dimethyl sulfoxide - FBS fetal bovine serum - GM growth medium - MEM Eagle's minimum essential medium - MMS methylmethane sulfonate - MNNG N-Methyl-N-Nitro-N-Nitrosoguanidine - PDL population doubling - SCC squamous cell carcinoma     

Expression of microRNAs in cerebrospinal fluid of dogs with central nervous system disease

In this pilot study we investigated the expression of 14 microRNAs in the cerebrospinal fluid (CSF) of dogs with neoplastic, inflammatory and degenerative disorders affecting the central nervous system (CNS). CSF microRNA (miRNA) expression profiles were compared to those from dogs with neurological signs but no evidence of structural or inflammatory CNS disease. Seven miRNAs were easily detected in all samples: miR-10b-5p, miR-19b, miR-21-5p, miR-30b-5p, miR-103a-3p, miR-124, and miR-128-3p. Expression of miR-10b-5p was significantly higher in the neoplastic group compared to other groups. There was no relation between miRNA expression and either CSF nucleated cell count or CSF protein content. Higher expression of miR-10b-5p in the neoplastic group is consistent with previous reports in human medicine where aberrant expression of miR-10b is associated with various neoplastic diseases of the CNS.     

The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities

MicroRNAs (miRNAs) are noncoding small RNAs that repress protein translation by targeting specific messenger RNAs. miR-15a and miR-16-1 act as putative tumor suppressors by targeting the oncogene BCL2. These miRNAs form a cluster at the chromosomal region 13q14, which is frequently deleted in cancer. Here, we report that the miR-15a and miR-16-1 cluster targets CCND1 (encoding cyclin D1) and WNT3A, which promotes several tumorigenic features such as survival, proliferation and invasion. In cancer cells of advanced prostate tumors, the miR-15a and miR-16 level is significantly decreased, whereas the expression of BCL2, CCND1 and WNT3A is inversely upregulated. Delivery of antagomirs specific for miR-15a and miR-16 to normal mouse prostate results in marked hyperplasia, and knockdown of miR-15a and miR-16 promotes survival, proliferation and invasiveness of untransformed prostate cells, which become tumorigenic in immunodeficient NOD-SCID mice. Conversely, reconstitution of miR-15a and miR-16-1 expression results in growth arrest, apoptosis and marked regression of prostate tumor xenografts. Altogether, we propose that miR-15a and miR-16 act as tumor suppressor genes in prostate cancer through the control of cell survival, proliferation and invasion. These findings have therapeutic implications and may be exploited for future treatment of prostate cancer.     

M3 subtype of muscarinic acetylcholine receptor promotes cardioprotection via the suppression of miR-376b-5p

The M(3) subtype of muscarinic acetylcholine receptors (M(3)-mAChR) plays a protective role in myocardial ischemia and microRNAs (miRNAs) participate in many cardiac pathophysiological processes, including ischemia-induced cardiac injury. However, the role of miRNAs in M(3)-mAChR mediated cardioprotection remains unexplored. The present study was designed to identify miRNAs that are involved in cardioprotective effects of M(3)-mAChR against myocardial ischemia and elucidate the underlying mechanisms. We established rat model of myocardial ischemia and performed miRNA microarray analysis to identify miRNAs involved in the cardioprotection of M(3)-mAChR. In H9c2 cells, the viability, intracellular free Ca(2+) concentration ([Ca(2+)]i), intracellular reactive oxygen species (ROS), miR-376b-5p expression level, brain derived neurophic factor (BDNF) and nuclear factor kappa-B (NF-κB) levels were measured. Our results demonstrated that M(3)-mAChR protected myocardial ischemia injury. Microarray analysis and qRT-PCR revealed that miR-376b-5p was significantly up-regulated in ischemic heart tissue and the M(3)-mAChRs agonist choline reversed its up-regulation. In vitro, miR-376b-5p promoted H(2)O(2)-induced H9c2 cell injuries measured by cells viability, [Ca(2+)]i and ROS. Western blot and luciferase assay identified BDNF as a direct target of miR-376b-5p. M(3)-mAChR activated NF-κB and thereby inhibited miR-376b-5p expression. Our data show that a novel M(3)-mAChR/NF-κB/miR-376b-5p/BDNF axis plays an important role in modulating cardioprotection. MiR-376b-5p promotes myocardial ischemia injury possibly by inhibiting BDNF expression and M(3)-mAChR provides cardioprotection at least partially mediated by the downregulation of miR-376b-5p through NF-κB. These findings provide new insight into the potential mechanism by which M(3)-mAChR provides cardioprotection against myocardial ischemia injury.     

Overlapping expression of microRNAs in human embryonic colon and colorectal cancer

MicroRNAs (miRNAs) are essential for regulating cell differentiation and maintaining the pluripotent state of stem cells. Although dysregulation of specific miRNAs has been associated with certain types of cancer, to date no evidence has linked miRNA expression in embryonic and tumor tissues. We assessed the expression of mature miRNAs in human embryonic colon tissue, and in colorectal cancer and paired normal colon tissue. Overlapping miRNA expression was detected between embryonic colonic mucosa and colorectal cancer. We have found that the miR-17-92 cluster and its target, E2F1, exhibit a similar pattern of expression in human colon development and colonic carcinogenesis, regulating cell proliferation in both cases. In situ hybridization confirmed the high level of expression of miR-17-5p in the crypt progenitor compartment. We conclude that miRNA pathways play a major role in both embryonic development and neoplastic transformation of the colonic epithelium.     

Two microRNAs, miR-330 and miR-125b-5p, mark the juxtaglomerular cell and balance its smooth muscle phenotype

We have shown that microRNAs (miRNAs) are necessary for renin cell specification and kidney vascular development. Here, we used a screening strategy involving microarray and in silico analyses, along with in situ hybridization and in vitro functional assays to identify miRNAs important for renin cell identity. Microarray studies using vascular smooth muscle cells (SMCs) of the renin lineage and kidney cortex under normal conditions and after reacquisition of the renin phenotype revealed that of 599 miRNAs, 192 were expressed in SMCs and 234 in kidney cortex. In silico analysis showed that the highly conserved miR-330 and miR-125b-5p have potential binding sites in smoothelin (Smtn), calbindin 1, smooth muscle myosin heavy chain, α-smooth muscle actin, and renin genes important for the myoepithelioid phenotype of the renin cell. RT-PCR studies confirmed miR-330 and miR-125b-5p expression in kidney and SMCs. In situ hybridization revealed that under normal conditions, miR-125b-5p was expressed in arteriolar SMCs and in juxtaglomerular (JG) cells. Under conditions that induce reacquisition of the renin phenotype, miR-125b-5p was downregulated in arteriolar SMCs but remained expressed in JG cells. miR-330, normally absent, was expressed exclusively in JG cells of treated mice. In vitro functional studies showed that overexpression of miR-330 inhibited Smtn expression in SMCs. On the other hand, miR-125b-5p increased Smtn expression, whereas its inhibition reduced Smtn expression. Our results demonstrate that miR-330 and miR-125b-5p are markers of JG cells and have opposite effects on renin lineage cells: one inhibiting and the other favoring their smooth muscle phenotype.     

MicroRNA expression profile of colon cancer stem-like cells in HT29 adenocarcinoma cell line

Increasing evidence has suggested cancer stem cells (CSCs) are considered to be responsible for cancer formation, recurrence, and metastasis. Recently, many studies have also revealed that microRNAs (miRNAs) strongly implicate in regulating self renewal and tumorigenicity of CSCs in human cancers. However, with respect to colon cancer, the role of miRNAs in stemness maintenance and tumorigenicity of CSCs still remains to be unknown. In the present study, we isolated a population of colon CSCs expressing a CD133 surface phenotype from human HT29 colonic adenocarcinoma cell line by Flow Cytometry Cell Sorting. The CD133⁺ cells possess a greater tumor sphere-forming efficiency in vitro and higher tumorigenic potential in vivo. Furthermore, the CD133⁺ cells are endowed with stem/progenitor cells-like property including expression of “stemness” genes involved in Wnt2, BMI1, Oct3/4, Notch1, C-myc and other genes as well as self-renewal and differentiation capacity. Moreover, we investigated the miRNA expression profile of colon CSCs using miRNA array. Consequently, we identified a colon CSCs miRNA signature comprising 11 overexpressed and 8 underexpressed miRNAs, such as miR-429, miR-155, and miR-320d, some of which may be involved in regulation of stem cell differentiation. Our results suggest that miRNAs might play important roles in stemness maintenance of colon CSCs, and analysis of specific miRNA expression signatures may contribute to potential cancer therapy.     

Opposing actions of the progesterone metabolites, 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP) on mitosis,apoptosis, and expression of Bcl-2, Bax and p21 in human breast cell lines

Previous studies have shown that breast tissues and breast cell lines convert progesterone (P) to 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP) and that 3αHP suppresses, whereas 5αP promotes, cell proliferation and detachment. The objectives of the current studies were to determine if the 5αP- and 3αHP-induced changes in cell numbers are due to altered rates of mitosis and/or apoptosis, and if 3αHP and 5αP act on tumorigenic and non-tumorigenic cells, regardless of estrogen (E) and P receptor status. The studies were conducted on tumorigenic (MCF-7, MDA-MB-231, T47D) and non-tumorigenic (MCF-10A) human breast cell lines, employing several methods to assess the effects of the hormones on cell proliferation, mitosis, apoptosis and expression of Bcl-2, Bax and p21. In all four cell lines, 5αP increased, whereas 3αHP decreased cell numbers, [³H]thymidine uptake and mitotic index. Apoptosis was stimulated by 3αHP and suppressed by 5αP. 5αP resulted in increases in Bcl-2/Bax ratio, indicating decreased apoptosis; 3αHP resulted in decreases in Bcl-2/Bax ratio, indicating increased apoptosis. The effects of either 3αHP or 5αP on cell numbers, [³H]thymidine uptake, mitosis, apoptosis, and Bcl-2/Bax ratio, were abrogated when cells were treated simultaneously with both hormones. The expression of p21 was increased by 3αHP, and was unaffected by 5αP. The results provide the first evidence that 5αP stimulates mitosis and suppresses apoptosis, whereas 3αHP inhibits mitosis and stimulates apoptosis. The opposing effects of 5αP and 3αHP were observed in all four breast cell lines examined and the data suggest that all breast cancers (estrogen-responsive and unresponsive) might be suppressed by blocking 5αP formation and/or increasing 3αHP. The findings further support the hypothesis that progesterone metabolites are key regulatory hormones and that changes in their relative concentrations in the breast microenvironment determine whether breast tissues remain normal or become cancerous.     

Large oncosomes mediate intercellular transfer of functional microRNA

Prostate cancer cells release atypically large extracellular vesicles (EVs), termed large oncosomes, which may play a role in the tumor microenvironment by transporting bioactive molecules across tissue spaces and through the blood stream. In this study, we applied a novel method for selective isolation of large oncosomes applicable to human platelet-poor plasma, where the presence of caveolin-1-positive large oncosomes identified patients with metastatic disease. This procedure was also used to validate results of a miRNA array performed on heterogeneous populations of EVs isolated from tumorigenic RWPE-2 prostate cells and from isogenic non-tumorigenic RWPE-1 cells. The results showed that distinct classes of miRNAs are expressed at higher levels in EVs derived from the tumorigenic cells in comparison to their non-tumorigenic counterpart. Large oncosomes enhanced migration of cancer-associated fibroblasts (CAFs), an effect that was increased by miR-1227, a miRNA abundant in large oncosomes produced by RWPE-2 cells. Our findings suggest that large oncosomes in the circulation report metastatic disease in patients with prostate cancer, and that this class of EV harbors functional molecules that may play a role in conditioning the tumor microenvironment.