RNA-based applications requiring high quality, non-degraded RNA are a foundational element of many research studies. As such, it is paramount that the integrity of experimental RNA is validated prior to cDNA synthesis or other downstream applications. In the absence of expensive equipment such as microfluidic electrophoretic devices, and as an alternative to the costly and time-consuming standard formaldehyde gel, RNA quality can be quickly analyzed by adding small amounts of commercial bleach to TAE buffer-based agarose gels prior to electrophoresis. In the presence of low concentrations of bleach, the secondary structure of RNA is denatured and potential contaminating RNases are destroyed. Because of this, the ‘bleach gel’ is a functional approach that addresses the need for an inexpensive and safe way to evaluate RNA integrity and will improve the ability of researchers to rapidly analyze RNA quality.
Agarose; Bleach; Denaturing gel; Electrophoresis; RNA quality
Oncostatin M (OSM) is an interleukin-6 (IL-6) family cytokine that has been implicated in a number of biological processes including inflammation, hematopoiesis, immune responses, development, and bone homeostasis. Recent evidence suggests that OSM may promote breast tumor invasion and metastasis. We investigated the role of OSM in the formation of bone metastases in vivo using the 4T1.2 mouse mammary tumor model in which OSM expression was knocked down using shRNA (4T1.2-OSM). 4T1.2-OSM cells were injected orthotopically into Balb/c mice, resulting in a greater than 97% decrease in spontaneous metastasis to bone compared to control cells. Intratibial injection of these same 4T1.2-OSM cells also dramatically reduced the osteolytic destruction of trabecular bone volume compared to control cells. Furthermore, in a tumor resection model, mice bearing 4T1.2-OSM tumors showed an increase in survival by a median of 10 days. To investigate the specific cellular mechanisms important for OSM-induced osteolytic metastasis to bone, an in vitro model was developed using the RAW 264.7 preosteoclast cell line co-cultured with 4T1.2 mouse mammary tumor cells. Treatment of co-cultures with OSM resulted in a 3-fold induction of osteoclastogenesis using the TRAP assay. We identified several tumor cell–induced factors including vascular endothelial growth factor, IL-6, and a previously uncharacterized OSM-regulated bone metastasis factor, amphiregulin (AREG), which increased osteoclast differentiation by 4.5-fold. In addition, pretreatment of co-cultures with an anti-AREG neutralizing antibody completely reversed OSM-induced osteoclastogenesis. Our results suggest that one mechanism for OSM-induced osteoclast differentiation is via an AREG autocrine loop, resulting in decreased osteoprotegerin secretion by the 4T1.2 cells. These data provide evidence that OSM might be an important therapeutic target for the prevention of breast cancer metastasis to bone.
oncostatin M; OSM; bone metastasis; breast cancer; osteolysis; osteoclastogenesis
Metastatic events to the bone occur frequently in numerous cancer types such as breast, prostate, lung, and renal carcinomas, melanoma, neuroblastoma, and multiple myeloma. Accumulating evidence suggests that the inflammatory cytokine interleukin (IL)-6 is frequently upregulated and is implicated in the ability of cancer cells to metastasize to bone. IL-6 is able to activate various cell signaling cascades that include the STAT (signal transducer and activator of transcription) pathway, the PI3K (phosphatidylinositol-3 kinase) pathway, and the MAPK (mitogen-activated protein kinase) pathway. Activation of these pathways may explain the ability of IL-6 to mediate various aspects of normal and pathogenic bone remodeling, inflammation, cell survival, proliferation, and pro-tumorigenic effects. This review article will discuss the role of IL-6: 1) in bone metabolism, 2) in cancer metastasis to bone, 3) in cancer prognosis, and 4) as potential therapies for metastatic bone cancer.
interleukin-6; bone metastasis; cancer; osteoclastogenesis; osteoclast; osteoblast
Existing prostate cancer cell lines have been derived from late stages of human prostate cancer. In this paper, we present two cell lines generated from prostatic intraepithelial neoplasia (PIN), the precursor lesion for prostate adenocarcinoma. Pr-111 and Pr-117 were established from PIN lesions that developed in the C3(1)/Tag transgenic model of prostate cancer. Pr-111 and Pr-117 cells express simian virus 40 large T antigen (SV40 Tag) and are immortalized in culture, distinguishing them from normal prostate cells. The growth rates of these two cell lines are quite different; with Pr-111 cells growing much more slowly (doubling time approximately 40 hours) compared to Pr-117 cells (doubling time approximately 22 hours), and also show significantly different growth rates in different media. Both prostate cell lines express cytokeratin and androgen receptor (AR) with Pr-111 cells demonstrating androgen-dependent growth and Pr-117 cells exhibiting androgen-responsive growth characteristics. Athymic nude mice injected with Pr-111 cells either do not develop tumors or develop tumors after a long latency period of 14 weeks. Pr-117 cells, however, develop tumors by 3 to 6 weeks, suggesting that Pr-117 cells represent a later stage of tumor progression. These two novel cell lines will be useful for studying early stages of prostate tumor development and androgen responsiveness.
prostate; PIN; adenocarcinoma; cell lines; mouse; AR, androgen receptor; BSA, bovine serum albumin; CS-FBS, charcoal-stripped fetal bovine serum; DHT, dihydrotestosterone; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GM, growth medium; H&E, hematoxylin and eosin; HPV, human papilloma virus; MTT, 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide; NDS, normal donkey serum; PIN, prostatic intraepithelial neoplasia; RT-PCR, reverse transcription-polymerase chain reaction; s.c., subcutaneous; SV40 Tag, simian virus 40 large T antigen
Tumor cell lines that can be tracked in vivo during tumorigenesis and metastasis provide vital tools for studying the specific cellular mechanisms that mediate these processes as well as investigating therapeutic targets to inhibit them. The goal of this study was to engineer imageable mouse mammary tumor cell lines with discrete propensities to metastasize to bone in vivo. Two novel luciferase expressing cell lines were developed and characterized for use in the study of breast cancer metastasis to bone in a syngeneic mouse model.
The 4 T1.2 luc3 and 66c14 luc2 cell lines were shown to have high levels of bioluminescence intensity in vitro and in vivo after orthotopic injection into mouse mammary fat pads. The 4 T1.2 luc3 cell line was found to closely model the sites of metastases seen in human patients including lung, liver, and bone. Specifically, 4 T1.2 luc3 cells demonstrated a high incidence of metastasis to spine, with an ex-vivo BLI intensity three orders of magnitude above the commercially available 4 T1 luc2 cells. 66c14 luc2 cells also demonstrated metastasis to spine, which was lower than that of 4 T1.2 luc3 cells but higher than 4 T1 luc2 cells, in addition to previously unreported metastases in the liver. High osteolytic activity of the 4 T1.2 luc3 cells in vivo in the bone microenvironment was also detected.
The engineered 4 T1.2 luc3 and 66c14 luc2 cell lines described in this study are valuable tools for studying the cellular events moderating the metastasis of breast tumor cells to bone.
Breast cancer; Mammary cancer; Bone metastasis; in vivo imaging; 4 T1 cells; 4 T1.2 cells; Osteolysis; Syngeneic Balb/c model
Little is known about collagen XI expression in normal and malignant breast tissue. Tissue microarrays, constructed from 72 patients with breast carcinoma and matched normal tissue, were immunohistochemically stained with five antisera against isoform-specific regions of collagen α1(XI) N-terminal domain. Staining intensity was graded on a 0–3 scale in epithelial cytoplasm, stroma, and endothelial staining of the vasculature of each tissue core. The staining was compared to known pathologic parameters: age, tumor size, overall tumor grade, nuclear grade, tubule formation, mitotic counts, angiolymphatic invasion, node status, estrogen receptor status, progesterone receptor status, and HER-2/neu status. Estrogen and progesterone receptor status were used as a control for comparison. With antisera V1a and amino propeptide (Npp), stroma surrounding cancerous cells was found to have decreased collagen α1(XI) staining compared to stroma adjacent to normal epithelium (P=0.0006, P<0.0001). Collagen α1(XI) staining with V1a antiserum in cytoplasm of cancer cells demonstrated decreased intensity in metastasized primary tumors when compared to nonmetastasized primary tumors (P=0.009). Cytoplasmic staining with Npp antiserum in cancer demonstrated an inverse relationship to positive estrogen receptor status in cancer (P=0.012) and to progesterone receptor status (P=0.044). Stromal staining for Npp in cancerous tissue demonstrated an inverse relationship with tubule formation score (P=0.015). This is the first study to localize collagen XI within normal and malignant breast tissue. Collagen α1(XI) appears to be downregulated in stroma surrounding breast cancer. Detection of collagen XI in breast tissue may help predict women who have lymph node metastases.
collagen type XI; breast cancer; tissue microarray; immunohistochemistry; node status; metastasis