Cancer Research: Effects of Insulin-like Factor -2 (IGF-2), Collagen, and Fibronectin on the Proliferation and α5-Integrins Expression of the Rhabdomyosarcoma-derived (RD) Cell Line
Cancer Research: Effects of Insulin-like Factor -2 (IGF-2), Collagen, and Fibronectin on the Proliferation and α5-Integrins Expression of the Rhabdomyosarcoma-derived (RD) Cell Line
DNA construct instability in bacteria used for Agrobacterium mediated plant t...iosrjce
The use of plasmid in the production of genetically modified (GM) crops is highly essential in
research and in commercial production of GM plants. However plasmid instability constitutes a major problem
in the use of recombined microorganisms in the production of GM crops. In this study we evaluated the stability
of p8114 carrying a gene coding for a transcription factor (TFIIIA) driven by Cassava Vein Mosaic Virus
(CsVMV) promoter and an nptII selectable marker driven by 35S promoter in the T-DNA. The plasmid was
amplified in E.coliDH5α strain on Luria Broth (LB)agar supplemented with 100 µg/ml kanamycin. The colonies
were confirmed by Restriction Fragment Length Analysis (RFLA) and by DNA sequencing. The confirmed
colonies were stored as glycerol stock at -80
0C and as DNA extracts in TE buffer at 40C. Agrobacterium strains
LBA4404, EHA 105 and AGL1 were also transformed with DNA from the confirmed colonies. Plasmid stability
was evaluated after 3 months. Sixteen to hundred percent level of instability was observed in E.colicolonies
stored at -80
0C and 50% level of instability in plasmid transformed into Agrobacterium strain LBA4404.
Agrobacterium strain LBA4404 showed a higher level of stability 75% compared to EHA 105 (0%) and AGL1 (50%).
Targeted Induced Local Lesions IN Genome. Mutations (Single base pair substitution) are created by traditionally used chemical mutagens. Identify SNPs and / or INDELS in a gene / genes of interest from a mutagenized population.
DNA construct instability in bacteria used for Agrobacterium mediated plant t...iosrjce
The use of plasmid in the production of genetically modified (GM) crops is highly essential in
research and in commercial production of GM plants. However plasmid instability constitutes a major problem
in the use of recombined microorganisms in the production of GM crops. In this study we evaluated the stability
of p8114 carrying a gene coding for a transcription factor (TFIIIA) driven by Cassava Vein Mosaic Virus
(CsVMV) promoter and an nptII selectable marker driven by 35S promoter in the T-DNA. The plasmid was
amplified in E.coliDH5α strain on Luria Broth (LB)agar supplemented with 100 µg/ml kanamycin. The colonies
were confirmed by Restriction Fragment Length Analysis (RFLA) and by DNA sequencing. The confirmed
colonies were stored as glycerol stock at -80
0C and as DNA extracts in TE buffer at 40C. Agrobacterium strains
LBA4404, EHA 105 and AGL1 were also transformed with DNA from the confirmed colonies. Plasmid stability
was evaluated after 3 months. Sixteen to hundred percent level of instability was observed in E.colicolonies
stored at -80
0C and 50% level of instability in plasmid transformed into Agrobacterium strain LBA4404.
Agrobacterium strain LBA4404 showed a higher level of stability 75% compared to EHA 105 (0%) and AGL1 (50%).
Targeted Induced Local Lesions IN Genome. Mutations (Single base pair substitution) are created by traditionally used chemical mutagens. Identify SNPs and / or INDELS in a gene / genes of interest from a mutagenized population.
Improved vector design eases cell line development workflow in the CHOZN GS-/...Merck Life Sciences
This poster was presented at ESACT meeting in 2017 in Lausanne, Switzerland. Cell line development for production of monoclonal antibody therapeutics requires an expression vector encoding both the heavy and light chains of the antibody. When expression of the heavy and lights chains is driven by the same promoter, the sequence redundancy can be problematic for verifying the vector sequence, copy number and insertion site in the host cell genome. This poster describes the work done to identify an expression vector that maintains a high level of antibody expression but lacks the sequence similarities, easing the cell line development workflow.
Epigenetics and it's relevance in crop improvementShamlyGupta
Epigenetics means ‘above’ or ‘on top of genetics’
A study of the changes in gene expression that are mitotically and/or meiotically heritable and do not involve a change in the DNA sequence
Gene-regulatory information that is not expressed in DNA sequences but transmitted from one generation (of cells or organisms) to the next
Coined by embryologist C. H. Waddington in 1942.
To handle complex Traits like Yield, different stress we must do modification in DNA molecular breeding techniques help us to do such changes in DNA to archive the Goals.
Targeting Induced Local Lesions IN Genomes (TILLING) is a combined tool of plant mutagenesis and DNA Biology to investigate useful mutations at Genomic level. First time used for cotton improvement.
Improved vector design eases cell line development workflow in the CHOZN GS-/...Merck Life Sciences
This poster was presented at ESACT meeting in 2017 in Lausanne, Switzerland. Cell line development for production of monoclonal antibody therapeutics requires an expression vector encoding both the heavy and light chains of the antibody. When expression of the heavy and lights chains is driven by the same promoter, the sequence redundancy can be problematic for verifying the vector sequence, copy number and insertion site in the host cell genome. This poster describes the work done to identify an expression vector that maintains a high level of antibody expression but lacks the sequence similarities, easing the cell line development workflow.
Epigenetics and it's relevance in crop improvementShamlyGupta
Epigenetics means ‘above’ or ‘on top of genetics’
A study of the changes in gene expression that are mitotically and/or meiotically heritable and do not involve a change in the DNA sequence
Gene-regulatory information that is not expressed in DNA sequences but transmitted from one generation (of cells or organisms) to the next
Coined by embryologist C. H. Waddington in 1942.
To handle complex Traits like Yield, different stress we must do modification in DNA molecular breeding techniques help us to do such changes in DNA to archive the Goals.
Targeting Induced Local Lesions IN Genomes (TILLING) is a combined tool of plant mutagenesis and DNA Biology to investigate useful mutations at Genomic level. First time used for cotton improvement.
The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and offering a wide range of dental certified courses in different formats.
Indian dental academy provides dental crown & Bridge,rotary endodontics,fixed orthodontics,
Dental implants courses.for details pls visit www.indiandentalacademy.com ,or call
0091-9248678078
Similar to Cancer Research: Effects of Insulin-like Factor -2 (IGF-2), Collagen, and Fibronectin on the Proliferation and α5-Integrins Expression of the Rhabdomyosarcoma-derived (RD) Cell Line
Cloning and expression of Human glutamic acid decarboxylase (GAD 65) gene in ...Open Access Research Paper
Diabetes is a chronic autoimmune disease characterized by the inability of body to produce or respond to insulin a hormone required by body to burn glucose for energy. Type I Diabetes mellitus, also known as Insulin Dependent Diabetes mellitus is a most frequent chronic disease of childhood, afflicts 0.2-0.3% of human individuals due to auto immune destruction of insulin secreting pancreatic β cells. GAD65 is the major auto antigen in Insulin Dependent Diabetes Mellitus (IIDM). Thus, this project is aimed at expression of GAD65 in E. coli. GAD65 gene was cloned into pET-28a bacterial expression vector and expression was studied in BL21 DE3 cells. Different parameters of induction like isopropyl-β-D-thiogalactopyranoside (IPTG), temperature, time interval were standardized. The recombinant clones induced with 2 μM of IPTG at 30oC for 4 h at flask level produced the protein upto 537μg/ml. Furthermore, the specificity of the purified recombinant protein was confirmed by western blot analysis using monoclonal antibodies. This work establishes a strategy in E. coli for the expression of GAD65 with optimized parameters.
The Cytotoxic and Antimigratory Activity of Brazilin-Doxorubicin on MCF-7/HER...UniversitasGadjahMada
Breast cancer cells with overexpression of HER2 are known to be more aggressive, invasive, and resistant to chemotherapeutic agent. Brazilin, the major compound in the Caesalpinia sappan L. (CS) heartwood, has been studied for it's anticancer activity. The purpose of this study was to investigate the cytotoxic and antimigratory activity of brazilin (Bi) in combination with doxorubicin (Dox) on MCF-7/HER2 cells. Cytotoxic activities of Bi individually and in combination with Dox were examined by MTT assay. Synergistic effects were analyzed by combination index (CI). Apoptosis and cell cycle profiles were observed by using flow cytometry. Migrating and invading cells were observed by using a Boyden chamber assay. Levels of MMP2 and MMP9 activity were observed by using a gelatin zymography assay. Levels of HER2, Bcl-2, Rac1, and p120 protein expression were observed by using an immunoblotting assay. The results of the MTT assay showed that Bi inhibited MCF-7/HER2 cell growth in a dose-dependent manner with an IC50 of 54 ± 3.7 μM. Furthermore, the combination of Bi and Dox showed a synergistic effect (CI <1). Flow cytometric analysis of Bi and its combination with Dox showed cellular accumulation in the G2/M phase and induction of apoptosis through suppression of Bcl-2 protein expression. In the Boyden chamber assay, gelatin zymography, and subsequent immunoblotting assay, the combination Bi and Dox inhibited migration, possibly through down regulation of MMP9, MMP2, HER2, Rac1, and p120 protein expression. Hence, it was deduced that Bi enhanced cytotoxic activity of Dox and inhibited migration of MCF-7/HER2 cells. Therefore, it is believed that it has strong potential to be developed for the treatment of metastatic breast cancer with HER2 overexpression.
LncRNA WARS2-IT1 Functions as an Oncogene and is Associated with Poor Outcome...semualkaira
Glioblastoma multiforme (GBM) is one of the most common malignant brain tumors in adults and has high mortality and relapse rates. Over the past few years, great advances have been made in the diagnosis and treatment of GBM, but unfortunately, the five-year overall survival rate of GBM patients is approximately 5.1%. Our study aimed to investigate the new mechanism of Long noncoding RNAs (lncRNAs) WARS2-IT1 regulate the malignant progression of Glioblastoma.
LncRNA WARS2-IT1 Functions as an Oncogene and is Associated with Poor Outcome...semualkaira
Glioblastoma multiforme (GBM) is one of the most common malignant brain tumors in adults and has high mortality and relapse rates. Over the past few years, great advances have been made in the diagnosis and treatment of GBM, but unfortunately, the five-year overall survival rate of GBM patients is approximately 5.1%. Our study aimed to investigate the new mechanism of Long noncoding RNAs (lncRNAs) WARS2-IT1 regulate the malignant progression of Glioblastoma.
LncRNA WARS2-IT1 Functions as an Oncogene and is Associated with Poor Outcome...semualkaira
Glioblastoma multiforme (GBM) is one of the most common malignant brain tumors in adults and has high mortality and relapse rates. Over the past few years, great advances have been made in the diagnosis and treatment of GBM, but unfortunately, the five-year overall survival rate of GBM patients is approximately 5.1%. Our study aimed to investigate the new mechanism of Long noncoding RNAs (lncRNAs) WARS2-IT1 regulate the malignant progression of Glioblastoma.
Effectiveness of Resveratrol on Metastasis: A Reviewiosrphr_editor
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Comparing Residual Integration Levels of Some IntegrationDeficient Lentiviral...inventionjournals
Lentiviral vectors (LVs) have many advantageous characteristics making them a good choice in the field of gene therapy. Nevertheless, their integration may lead to detrimental effects. To overcome this problem, lentiviral integration can be targeted through using integration-deficient lentiviral vectors (IDLVs). In this study, an integration-proficient lentiviral vector (IPLV) and a battery of IDLVs with single or multiple mutations affecting integration were produced and their integration levels were compared. eGFP time-course experiment and clonogenic assay were used to make these comparisons. It was found that there was not any significant difference between the residual integration of any of the IDLVs used in this study and that of the standard IDLV; D64V-IDLV. It can be concluded that most IDLV integration is mediated by integraseindependent mechanisms.
Similar to Cancer Research: Effects of Insulin-like Factor -2 (IGF-2), Collagen, and Fibronectin on the Proliferation and α5-Integrins Expression of the Rhabdomyosarcoma-derived (RD) Cell Line (20)
Implantation of a Tissue-engineered Heart Valve from Human Fibroblasts Exhibi...Raul Soto
Cardiovascular Tissue Engineering and Repair: Implantation of a Tissue-engineered Heart Valve from Human Fibroblasts Exhibiting Short Term Function in the Sheep Pulmonary Artery
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Cancer Research: Effects of Insulin-like Factor -2 (IGF-2), Collagen, and Fibronectin on the Proliferation and α5-Integrins Expression of the Rhabdomyosarcoma-derived (RD) Cell Line
1. IGF-2
(ng/m
l)IGF-2
(ng/m
l)
In this experiment we examined the proliferation and 5- integrin surface expression of the rhabdomyosarcoma (RD) cell line when cultured on collagen and fibronectin substrates. We also examined the impact of insulin-likeα
growth factor 2 (IGF-2) on proliferation and 5- integrin surface expression. After culturing RD cells for 0, 3, and 7 days on collagen and fibronectin 2D matrices at concentrations of 5µg/mL and 10µg/mL per condition, ourα
results indicate that the 10µg/mL fibronectin matrix facilitated the optimal proliferation. Interestingly, using FACS analysis, we identified a 20X increase in 5- integrin surface expression when cultured in both collagen andα
fibronectin when compared to their respective controls.
We continued our research by studying the effect of IGF-2 on RD cell proliferation and 5- integrin expression using a concentration of 100ng/mL. We found that there was no statistically significant impact on proliferation orα
5-integrin expression when compared to their respective controls. This leads us to believe there is a positive effect on 5- integrin surface expression considering when cultured with collagen and fibronectin alone, there is aα α
decrease in surface expression.
Confocal microscopy showed rapid and robust proliferation from day 0 to day 3 and day 7. Cell morphology was consistent with the material sheets being elongated and mutlinucleated. Fluorescence was uneven in several
photos which we hypothesize to be attributed to an insufficient amount of Live/Dead stain to the amount of RD cells per culture. Future experiments should be performed to confirm and optimize this issue. We noticed
standard deviations varying greatly when incubated with this stain and placed into a plate reader. Further optimization assays will be conducted in the future to address these issues.
Future experiments include culturing RD cells with various concentrations of IGF-2 on collagen and fibronectin substrates. We would then continue studying its proliferative effects as well as it’s impact on 5- integrin surfaceα
expression. We hope to expand this research by looking at overall integrin expression as indicated by ß1- integrin antibodies.
Effects of Insulin-like Factor -2 (IGF-2), Collagen, and Fibronectin on the ProliferationEffects of Insulin-like Factor -2 (IGF-2), Collagen, and Fibronectin on the Proliferation
andand αα5-Integrins Expression of the5-Integrins Expression of the Rhabdomyosarcoma-derived (RD) Cell LineRhabdomyosarcoma-derived (RD) Cell Line
Burke, K.*, Siu, V.*, Soto-Velez, R. * • Tawil, B, PhD, Instructor • Biomedical Engineering 502 : SUMMER 2012Burke, K.*, Siu, V.*, Soto-Velez, R. * • Tawil, B, PhD, Instructor • Biomedical Engineering 502 : SUMMER 2012
* California State University Channel Islands • One University Drive • Camarillo, CA 93012* California State University Channel Islands • One University Drive • Camarillo, CA 93012
RD cells (from ATCC) cultured in RD media (DMEM w/ 10% FBS), washed with sterile Phosphate Buffered Saline (PBS) and
trypsinized for 5 minutes at room temp (RT), post media-aspiration, were centrifuged (200rcf for 5 minutes) prior to
resuspension of cell pellet, in 1 mL of RD media. Cell concentrations were calculated using measurements obtained from the
Nexcelom T4 Cellometer. Incubation of RD cells onto 24-well plates were then performed first in 2D, then 3D, collagen and
fibronectin (SIGMA ALDRICH) matrices. Incubation (37°C) was maintained for Days 0, 3, and 7. Proliferation was measured
utilizing 150µL of live/dead Cell Viability assay (Invitrogen) added to each well and incubated for 20 minutes at RT. Confocal
images were obtained using an Olympus IX-71 Inverted Microscope and QCapture pro software as well as reading average
fluorescence intensity (494 nm ex / 517 nm em) over each well using a Molecular Devices F5 plate reader. FilterMax F5
Multi-mode Plate Reader Software (Molecular Devices) provided raw fluorescent read data. Raw data were analyzed and
evaluated using Microsoft Excel. 300µL of thrombin was then added to each well for 3D matrices. 400uL of RD media was
added to each well after 30 minutes, for a total amount of 1mL per well. -5 integrin expression was initiated with RD cellα
culture in collagen and fibronectin, incubated in 5mL 0.25% trypsin-EDTA for 5min at RT, neutralized with RD media,
centrifuged and decanted, with the pellet resuspended in RD media to 4x104
/mL cell stock. 500uL of cell stock used to seed
wells in 24-well plate to 2x102
cells/well, incubated for days 0 (1hr) and 7. Preparation for FACS analysis protocol (Guava
EasyCyte Mini flow cytometer) began with aspirating wells, post incubation, adding 500µL 0.25% trypsin-EDTA, incubating
for 5 min at RT and adding 500µL RD media, transferring cell solution to 1.5mL conical tube, and centrifuging. A single PBS
wash during centrifugation was performed prior to resuspending in 100uL anti 5α -antibody solution (1-10 dilution of anti α5
Ab in PBS) and incubating in ice for 60 min. Three total PBS washes during centrifugation were performed, with a final cell
pellet re-suspension in 300µL PBS. Experiments were repeated with the addition of IGF-2 to look at its effects on proliferation
and a5-integrin surface expression. The addition of IGF-2 (100 ng/mL) to RD cells within a 2D collagen matrix versus a 2D
fibronectin matrix, required five 24-well plates, each with collagen and fibronectin, measured at days 0, 3, & 7 for proliferation,
and days 0 and 7 for FACS. MiniTab 14 software was used produce histograms, 2-sample t-tests, and response-surface
computational models.
Materials and MethodsMaterials and Methods
Tumor metastasis, which involves the migration of cancer cells from the primary tumor to other organs, is responsible for a
majority of cancer-related deaths. Identification of the growth factors which promote cancer cell proliferation, and of the
integrins used by cancer cells for proliferation and motility, are key aspects in cancer research; as it is the first step in the
development of compounds with the ability to block the activity or inhibit the expression of these integrins and growth factors.
The purpose of this experiment is to determine if expression of 5- integrins is up-regulated in RD cells, and if IGF-2 has aα
significant effect on RD cells proliferation.
In some cancers, the interaction of 5- integrins with ECM proteins such as fibronectin are known to be involved inα
proliferation, migration, motility, and metastasis, and in helping cancer cells survive chemotherapy. Up-regulation of growth
factor signaling promotes cell proliferation and motility in soft tissue sarcomas, such as RMS. Insulin-like growth factor (IGF)-
family proteins are involved in the initiation and maintenance of the cancer phenotype in many types of adult and childhood
cancer. In particular, IGF-2 is overexpressed in RMS cells; and inhibition of its receptor IGF-2R with antibodies inhibits tumor
cell growth. RD is a human cell line derived from RMS. RD cells secrete IGF-2 endogenously as an autocrine factor to enable
proliferation and motility. This makes IGF-family proteins potential targets for anti-cancer therapies.
IntroductionIntroduction
AbstractAbstract
Rhabdomyosarcoma (RMS) is a rare type of soft tissue sarcoma, more common in children. In order to develop treatments, our
research aims to identify which integrins RMS cells use for motility, and which growth factors increase or decrease its
proliferation.
In this study, RD cells, derived from human RMS, were cultured in 2D fibronectin and collagen matrices. The effect of insulin-
like growth factor-2 (IGF-2) in RD cell proliferation was observed using fluorescent microscopy, and measured using a
Live/Dead stain. Response surface computational methods were used to construct a mathematical model of RD cell growth as a
function of the concentration of IGF-2, and the concentration of protein substrate. Expression of 5- integrins was measuredα
using flow cytometry using an anti- 5 antibody.α
Experimental results indicate that IGF-2 upregulates RD cell proliferation cultured in vitro in 2D fibronectin matrices, but not
when cultured in vitro in 2D collagen matrices. Results also show that the collagen concentration in the 2D matrix has a
significant and positive effect on RD cell proliferation, while the fibronectin concentration does not. Finally, it was found that
the expression of 5- integrins is up-regulated in RD cells treated with IGF-2α .
Figure 1. Effect of IGF-2 in RD Cell Proliferation for Various Collagen Constructs - (a) Experimental results show
that the presence of IGF-2 growth factor (100 ng/ml) did not have a significant effect on the proliferation of RD cells when
grown in 2D collagen constructs. Graph is standardized at 100% fluorescence against Day 0. Experimental results also
show that RD cell proliferation was greater at the lower concentration of collagen. (b) Response surface computational
model for RD cell proliferation, as a function of both collagen concentration and IGF-2 concentration.
Figure 4. Fluorescent micrographs depicting changes in cell
density as a function of collagen concentration and presence /
absence of IGF-2 growth factor, for three timepoints.
* = equal contributor
ResultsResults
ConclusionsConclusions
Figure 5. Fluorescent micrographs depicting changes in cell density as
a function of fibronectin concentration and presence / absence of IGF-
2 growth factor, for three timepoints.
Figure 2. Effect of IGF-2 in RD Cell Proliferation for Various Fibronectin Constructs - (a) Experimental results
show that the presence of IGF-2 growth factor (100 ng/ml) had a significant effect on the proliferation of RD cells
when grown in a 2D fibronectin construct. Graph is standardized at 100% fluorescence against Day 0. Experimental
results also show that the concentration of fibronectin did not have a statistically significant effect on RD cell
proliferation. (b) Response surface computational model for RD cell proliferation, as a function of both fibronectin
concentration and IGF-2 concentration.
(a) (b) (a) (b)
Figure 3. 5 integrin expression in RD cells treated with IGF-2 growth factor (100 ng/ml) increases after 7 days, inα
both collagen and fibronectin 2D constructs.
Experimental GroupsExperimental Groups
and Controlsand Controls
2D
RD cells in Collagen or Fibronectin 2D
matrix
Cells /well ConditionsConditions Sample typeSample type
1x104
Collagen 5µg/mL &
10µg/mL
EXPERIMENTAL
Fibronectin 5µg/mL
& 10µg/mL
No Collagen or
Fibronectin
CONTROL
0
Collagen or
Fibronectin
3D
Fibrinogen (F) + Thrombin (T) at various
concentrations in 3D matrix
Cells /well ConditionsConditions Sample typeSample type
2x104
F10+T20
EXPERIMENTAL
F10+T40
F20+T20
F20+T40
0
Same as above four
conditions
CONTROL
Integrin
Collagen and Fibronectin with the
addition of 5 integrin antibodyα
Cells /well ConditionsConditions Sample typeSample type
2x102
Collagen 5µg/mL
& 10µg/mL
EXPERIMENTAL
Fibronectin
5 and 10 µg/mL
No Collagen or
Fibronectin, with
Ab
CONTROL
No Collagen or
Fibronectin,
without Ab
IGF-2
Collagen and Fibronectin with the
addition of IGF-2 Growth Factor
Cells /well ConditionsConditions Sample typeSample type
2.5x104
Collagen 5µg/mL
& 10µg/mL
EXPERIMENTAL
Fibronectin 5 and
10 µg/mL
0
No Collagen or
Fibronectin without
IGF-II
CONTROL
Fibronectin
(µg/ml)
IGF-2 up-regulates expression of 5 integrinsα