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
PTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. CpG island hypermethylation is another route to tumour suppressor gene inactivation, however, the literature regarding PTEN hypermethylation in cancer is controversial. Furthermore, investigation of the methylation status of the PTEN CpG island is challenging due to sequence homology with the PTEN pseudogene, PTENP1. PTEN shares a CpG island promoter with another gene known as KLLN. Here we present a thorough reinvestigation of the methylation status of the PTEN CpG island in DNA from colorectal, breast, ovarian, glioma, lung and haematological cancer cell lines.
Using a range of bisulphite-based PCR assays we investigated 6 regions across the PTEN CpG island. We found that regions 1-4 were not methylated in cancer cell lines (0/36). By allelic bisulphite sequencing and pyrosequencing methylation was detected in regions 5 and 6 in colorectal, breast and haematological cancer cell lines. However, methylation detected in this region was associated with the PTENP1 promoter and not the PTEN CpG island.
We show that methylation of the PTEN CpG island is a rare event in cancer cell lines and that apparent methylation most likely originates from homologous regions of the PTENP1 pseudogene promoter. Future studies should utilize assays that reliably discriminate between PTEN and PTENP1 to avoid data misinterpretation.
DNA methylation; Epigenetic; PTEN; KILLIN; PTENP1; Pseudogene; Cowden syndrome
Traditional analyses of calcium homeostasis have separately quantified either calcium accumulation or release mechanisms. To define the system as a whole, however, requires multiple experimental techniques to examine both accumulation and release. Here we describe a technique that couples the simultaneous quantification of radio-labeled calcium accumulation in endoplasmic reticulum (ER) microsomes with the release of inorganic phosphate (Pi) by the hydrolytic activity of sarco-endoplasmic reticulum calcium ATPase (SERCA) all in the convenience of a 96-well format.
Calcium; SERCA activity; Microsomes; Inorganic phosphate; Malachite green
Quantifying tetrodotoxin (TTX) has been a challenge in both ecological and medical research due to the cost, time and training required of most quantification techniques. Here we present a modified Competitive Inhibition Enzymatic Immunoassay for the quantification of TTX, and to aid researchers in the optimization of this technique for widespread use with a high degree of accuracy and repeatability.
Tetrodotoxin; CIEIA; HPLC
Auscultation is an approach for diagnosing many cardiovascular problems. Automatic analysis of heartbeat sounds and extraction of its audio features can assist physicians towards diagnosing diseases. Textual transcription allows recording a continuous heart sound stream using a text format which can be stored in very small memory in comparison with other audio formats. In addition, a text-based data allows applying indexing and searching techniques to access to the critical events. Hence, the transcribed heartbeat sounds provides useful information to monitor the behavior of a patient for the long duration of time. This paper proposes a frequency shifting method in order to improve the performance of the transcription. The main objective of this study is to transfer the heartbeat sounds to the music domain. The proposed technique is tested with 100 samples which were recorded from different heart diseases categories. The observed results show that, the proposed shifting method significantly improves the performance of the transcription.
Dynamic chromatin structure is a fundamental property of gene transcriptional regulation, and has emerged as a critical modulator of physiological processes during cellular differentiation and development. Analysis of chromatin structure using molecular biology and biochemical assays in rare somatic stem and progenitor cells is key for understanding these processes but poses a great challenge because of their reliance on millions of cells. Through the development of a miniaturized genome-scale chromatin immunoprecipitation method (miniChIP–chip), we have documented the genome-wide chromatin states of low abundant populations that comprise hematopoietic stem cells and immediate progeny residing in murine bone marrow. In this report, we describe the miniChIP methodology that can be used for increasing an understanding of the epigenetic mechanisms underlying hematopoietic stem and progenitor cell function. Application of this method will reveal the contribution of dynamic chromatin structure in regulating the function of other somatic stem cell populations, and how this process becomes perturbed in pathological conditions.
Miniaturized chromatin immunoprecipitation assays; Microarray technology; Histone modifications; Stem and progenitor cells; Epigenetic regulation; Lineage commitment
We developed a rapid mutagenesis method based on a modification of the QuikChange® system (Stratagene) to systemically replace endogenous gene sequences with a unique similar size sequence tag. The modifications are as follows: 1: the length of the anchoring homologous sequences of both mutagenesis primers were increased to 16 - 22 bp to achieve melting temperatures greater than 80°C. 2: the final concentrations of both primers were increased to 5-10 ng/μl and the final concentration of template to 1-2 ng/μl. 3: the annealing temperature was adjusted when necessary from 52°C to 58°C. We generated 25 sequential mutants in the cloned espD gene (1.2 kb), which encodes an essential component of the type III secretion translocon required for the pathogenesis of enteropathogenic E. coli (EPEC) infection. Each mutation consisted of the replacement of 15 codons (45 bp) with 8 codons representing a 24 bp sequence containing three unique restriction endonuclease sites (KpnI/MfeI/SpeI) starting from the second codon. The insertion of the restriction endonuclease sites provides a convenient method for further insertions of purification and/or epitope tags into permissive domains. This method is rapid, site-directed and allows for the systematic creation of mutants evenly distributed throughout the entire gene of interest.
Mutagenesis, Site-Directed; Polymerase Chain Reaction; Plasmids; Sequence Deletion
Cell culturing, and the requisite storage of cell lines at ultra-low temperatures, is used in most laboratories studying or using eukaryotic proteomics, genomics, microarray, and RNA technologies. In this study we have observed that A72(dog), CRFK(cat), NB324K(human), MCF7(human), WI38(human), and C636(mosquito) cells were effectively cryopreserved at -80°C while attached to the substratum of 25cm2 tissue culture flasks. This was accomplished using a serum free crypreservative recently developed by Corsini and co-workers. The technique allows for significant savings of time and money in laboratories that rapidly process numerous cell lines.
Cryopreservation; Culture Media, Serum-Free; Cells, Cultured
Guanine-rich RNA oligonucleotides display many novel structural motifs in recent crystal structures. Here we describe the procedures of the chemical synthesis and the purification of such RNA molecules that are suitable for X-ray crystallographic studies. Modifications of the previous purification methods allow us to obtain better yields in shorter time. We also provide 24 screening conditions that are very effective in crystallization of the guanine-rich RNA oligonucleotides. Optimal crystallization conditions are usually achieved by adjustment of the concentration of the metal ions and pH of the buffer. Crystals obtained by this method usually diffract to high resolution.
Isolation and Purification; Crystallization; Oligonucleotides
Elucidating the mechanism of over and under expression of proteins is critical in developing a better understanding of cancer. Multiple techniques are used to examine differential expression of proteins in cells and assess changes in protein expression in response to therapies such as radiation. Reduced expression can be caused by protein inactivation, mRNA instability, or reduced transcription. The following protocol was used to determine the mechanism for the reduced expression of an antiapoptotic factor, survivin, in normal tissues in response to radiation and the defect in cancer cells that prevents this reduction. We also examined ways to overcome survivin over expression in cancer cells in order to sensitize them to radiation. We will focus on the use of antisense oligonucleotides, cell cycle analysis, and luciferase reporter genes.
Survivin; Antisense oligonucleotides; Luciferase
Herein we have explored two practical aspects of cryopreserving cultured mammalian cells during routine laboratory maintenance. First, we have examined the possibility of using a serum-free, hence more affordable, cryopreservative. Using five mammalian lines (Crandell Feline Kidney, MCF7, A72, WI 38 and NB324K), we found that the serum-free alternative preserves nearly as efficiently as the serum-containing preservatives. Second, we compared cryostorage of those cells in suspended versus a pellet form using both aforementioned cryopreservatives. Under our conditions, cells were in general recovered equally well in a suspended versus a pellet form.
Cryopreservation; Cells, Cultured; Culture Media, Serum-Free
Experimental modulation of capsule size is an important technique for the study of the virulence of the encapsulated pathogen Cryptococcus neoformans. In this paper, we summarize the techniques available for experimental modulation of capsule size in this yeast and describe improved methods to induce capsule size changes. The response of the yeast to the various stimuli is highly dependent on the cryptococcal strain. A high CO2 atmosphere and a low iron concentration have been used classically to increase capsule size. Unfortunately, these stimuli are not reliable for inducing capsular enlargement in all strains. Recently we have identified new and simpler conditions for inducing capsule enlargement that consistently elicited this effect. Specifically, we noted that mammalian serum or diluted Sabouraud broth in MOPS buffer pH 7.3 efficiently induced capsule growth. Media that slowed the growth rate of the yeast correlated with an increase in capsule size. Finally, we summarize the most commonly used media that induce capsule growth in C. neoformans.
Cryptococcus neoformans; Infection; Virulence
The discovery of oncogenes and signal transduction
pathways important for mitogenesis has triggered the development of
target-specific small molecule anti-cancer compounds. As exemplified by
imatinib (Gleevec), a specific inhibitor of the Chronic Myeloid Leukemia
(CML)-associated Bcr-Abl kinase, these agents promise impressive activity in
clinical trials, with low levels of clinical toxicity. However, such therapy is
susceptible to the emergence of drug resistance due to amino acid substitutions
in the target protein. Defining the spectrum of such mutations is important for
patient monitoring and the design of next-generation inhibitors. Using imatinib
and BCR/ABL as a paradigm for a drug-target pair, we recently reported a
retroviral vector-based screening strategy to identify the spectrum of
resistance-conferring mutations. Here we provide a detailed methodology for the
screen, which can be generally applied to any drug-target pair.
Genes, ABL; Chronic myeloid leukemia; Drug resistance
It is widely understood that tumor cells express tumor-associated antigens (TAAs), of which many are usually in low immunogenicity; for example, carcinoembryonic antigen (CEA) is specifically expressed on human colon cancer cells and is viewed as a low-immunogenic TAA. How to activate host immunity against specific TAAs and to suppress tumor growth therefore becomes important in cancer therapy development.
To enhance the immune efficiency of CEA in mice that received, we fused a partial CEA gene with exogenous SARS-CoV fragments. Oral vaccination of an attenuated Salmonella typhimurium strain transformed with plasmids encoding CEA-SARS-CoV fusion gene into BALB/c mice elicited significant increases in TNF-α and IL-10 in the serum. In addition, a smaller tumor volume was observed in CT26/CEA-bearing mice who received CEA-SARS-CoV gene therapy in comparison with those administered CEA alone.
The administration of fusing CEA-SARS-CoV fragments may provide a promising strategy for strengthening the anti-tumor efficacy against low-immunogenic endogenous tumor antigens.
immunotherapy; tumor-derived peptide; tumor vaccine; low-immunogenicity
The APCmin/+ mouse is commonly used in cancer research and is just one of many genetically altered models that is currently being developed. With high numbers of breeding programs, it is important to have a simple method that can be used to genotype the mice non-invasively. Here we report a reproducible method for genotyping mice with DNA extracted from fecal samples. Comparison of fecal results with those obtained from intestinal tissue DNA and clinical outcome (presence/absence of tumors) showed this technique to have 100% accuracy. This non-invasive method of genotyping may be applied to other transgenic mouse models.
APCmin/+; feces; genotyping; cancer; non-invasive
In the preparation of transgenic murine ES cells it is important to verify the construct has a single insertion, because an ectopic neomycin phosphortransferase positive selection cassette (NEO) may cause a position effect. During a recent work, where a knockin SCA28 mouse was prepared, we developed two assays based on Real-Time PCR using both SYBR Green and specific minor groove binder (MGB) probes to evaluate the copies of NEO using the comparative delta-delta Ct method versus the Rpp30 reference gene.
We compared the results from Southern blot, routinely used to quantify NEO copies, with the two Real-Time PCR assays. Twenty-two clones containing the single NEO copy showed values of 0.98 ± 0.24 (mean ± 2 S.D.), and were clearly distinguishable from clones with two or more NEO copies.
This method was found to be useful, easy, sensitive and fast and could substitute for the widely used, but laborious Southern blot method.
A cell-counting algorithm, developed in Matlab®, was created to efficiently count migrated fluorescently-stained cells on membranes from migration assays. At each concentration of cells used (10,000, and 100,000 cells), images were acquired at 2.5 ×, 5 ×, and 10 × objective magnifications. Automated cell counts strongly correlated to manual counts (r2 = 0.99, P < 0.0001 for a total of 47 images), with no difference in the measurements between methods under all conditions. We conclude that our automated method is accurate, more efficient, and void of variability and potential observer bias normally associated with manual counting.
automated cell counting; threshold; migration assays; manual cell counting
We describe here a method to rapidly convert any desirable DNA fragment, as small as 100 bp, into long tandem DNA arrays up to 140 kb in size that are inserted into a microbe vector. This method includes rolling-circle phi29 amplification (RCA) of the sequence in vitro and assembly of the RCA products in vivo by homologous recombination in the yeast Saccharomyces cerevisiae. The method was successfully used for a functional analysis of centromeric and pericentromeric repeats and construction of new vehicles for gene delivery to mammalian cells. The method may have general application in elucidating the role of tandem repeats in chromosome organization and dynamics. Each cycle of the protocol takes ~ two weeks to complete.
Although yeast two-hybrid experiments are commonly used to identify protein interactions, the frequent occurrence of false negatives and false positives hampers data interpretation. Using both yeast one-hybrid and two-hybrid experiments, we have identified potential sources of these problems: the media preparation protocol and the source of the yeast nitrogen base may not only impact signal range but also effect whether a result appears positive or negative. While altering media preparation may optimize signal differences for individual experiments, media preparation must be reported in detail to replicate studies and accurately compare results from different experiments.
Yeast one-hybrid; yeast two-hybrid; protein-protein interaction; accuracy; false positive; false negative
DNA isolation from C. neoformans is difficult due to a thick and resistant capsule. We have optimized a new and rapid DNA isolation method for Cryptococcus using a short urea treatment followed by a rapid method using a chelex resin suspension. This procedure is simpler than previously reported methods.
Rodent models have been used extensively to study mammary gland development and for studies of toxicology and carcinogenesis. Mammary gland gross morphology can visualized via the excision of intact mammary gland chains following fixation and staining with carmine using a tissue preparation referred to as a whole mount. Methods are described for the automated collection of digital images from an entire mammary gland whole mount and for the interrogation of digital data using a "masking" technique available with Image-Pro® plus image analysis software (Mediacybernetics. Silver Spring, MD).
Parallel to mammographic analysis in humans, measurements of rodent mammary gland density were derived from area-based or volume-based algorithms and included: total circumscribed mammary fat pad mass, mammary epithelial mass, and epithelium-free fat pad mass. These values permitted estimation of absolute mass of mammary epithelium as well as breast density. The biological plausibility of these measurements was evaluated in mammary whole mounts from rats and mice. During mammary gland development, absolute epithelial mass increased linearly without significant changes in mammographic density. Treatment of rodents with tamoxifen, 9-cis-retinoic acid, or ovariectomy, and occurrence of diet induced obesity decreased both absolute epithelial mass and mammographic density. The area and volumetric methods gave similar results.
Digital image analysis can be used for screening agents for potential impact on reproductive toxicity or carcinogenesis as well as for mechanistic studies, particularly for cumulative effects on mammary epithelial mass as well as translational studies of mechanisms that explain the relationship between epithelial mass and cancer risk.
We describe a simple and reproducible method to measure absolute telomere length (aTL) using quantitative real-time polymerase chain reaction (qPCR). This method is based on the Cawthon method for relative measurement of telomere length (TL) but modified by introducing an oligomer standard to measure aTL. The method describes the oligomer standards, the generation of the standard curve and the calculations required to calculate aTL from the qPCR data. The necessary controls and performance characteristics of the assay are described in detail and compared relative to other methods for measuring TL. Typical results for this assay for a variety of human tissue samples are provided as well as a troubleshooting schedule. This method allows high throughput measurement of aTL using small amounts of DNA making it amenable for molecular epidemiological studies. Compared to the traditional relative TL qPCR assays, the aTL method described in this protocol enables a more direct comparison of results between experiments within and between laboratories.
Real-time quantitative RT-PCR (RT-qPCR) is a "gold" standard for measuring steady state mRNA levels in RNA interference assays. The knockdown of the epidermal growth factor receptor (EGFR) gene with eight individual EGFR small interfering RNAs (siRNAs) was estimated by RT-qPCR using three different RT-qPCR primer sets.
Our results indicate that accurate measurement of siRNA efficacy by RT-qPCR requires careful attention for the selection of the primers used to amplify the target EGFR mRNA.
We conclude that when assessing siRNA efficacy with RT-qPCR, more than one primer set targeting different regions of the mRNA should be evaluated and at least one of these primer sets should amplify a region encompassing the siRNA recognition sequence.
NF-κB regulates several important expressions, such as cytokine release, anti-apoptosis, adhesion molecule expression, and cell cycle processing. Several NF-κB inhibitors have been discovered as an anti-tumor or anti-inflammatory drug. The activity of NF-κB transcription factor is negatively regulated by IκB binding. In this study, IκB assay system was established and IκB–EGFP fusion protein was used as an indicator to monitor the effects of substances on the IκB degradation. The results indicated that the chosen hydroquinone could inhibit the IκB degradation and cause the cell de-attachment from the bottom of culture plate. In addition, this system could also monitor the IκB degradation of microbial metabolite of natural mixtures of propolis. Thus, the IκB assay system may be a good system for drug discovery related to microbial metabolite.
Microbial metabolite; Antioxidant; IκB; EGFP; Hydroquinone; Propolis