"

From 2013.igem.org

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Its purified state has shown to induce apoptosis in infected thymic precursors, hemocytoblasts, chicken lymphoblastoid cells lines, malignant human lymphoblastoid cells, human osteosarcoma cells, and almost every other cells lines tested.

It is interesting to note that Apoptin selectively induces apoptosis in diverse cancer cells including hepatoma, osteosarcoma, melanoma, cholangiocarcinoma, colon carcinoma, lung cancer, breast cancer, prostate cancer, cervix cancer, gastric cancer, but has no effect on cell death of normal cells. It is because tumor cells are sensitive to Apoptin whereas the corresponding normal cells are resistant and a possible cause for Apoptin´s distinctive subcellular localization is two principal sequences including nuclear localization signals, NLSs and nuclear export signals, NESs. Apoptin has a distinct leucine rich nuclear export sequence (LR-NES) located at the N-terminus of the protein between amino acids 33-46 and that facilitates nuclear import. To use Apoptin in cancer therapy, it was necessary a secretory TAT-apoptin fusion protein by adding a secretory signal to the N-terminal of the recombinant molecule. The secreted TAT-apoptin enters to infected cells and causes apoptosis.

About the relation with the apoptosis´s pathways, the cell death caused by Apoptin is independent of p53 and the Bcl-2. Apoptin can induce apoptosis in human tumor cells without the involvement of p53, Bcl-2, belonging to a family of Bcl-2-like proteins, acts as an apoptosis inhibiter. However, the activity of Apoptin-induced apoptosis is even enhanced by overexpressing Bcl-2.
",s234="Future Work",s424="Results",s250="Internalization module relies on the ability of the HIV-1 trans-activator protein TAT protein transduction domain to deliver proteins across the cell membrane. Tat-fusion proteins internalization takes place in approximately 20 minutes and is dependent on the extracellular concentration of the Tat-fusion protein. (Vocero-Akbani et al., 1999).

Tat-Apoptin fusion protein was transfected with an efficiency of >90%. In normal cells it stays in the cytoplasm where it has no significant effect, on the other hand in cancer cells it migrates into the nucleus where it triggers apoptosis. (Guelen, L. et al., 2004).

The biobrick was designed in a way that the gene of interest is inserted in-frame at the 3’ end of Tat. This assures internalization of the protein of interest. Tat-GFP fusion protein was used as proof of concept for this biobrick. To perform the internalization assay His-TAT-GFP (TG) and His-GFP (HG) were used, purified via His-tag (HisPur, Thermo Scientific). Both constructs were cloned into pUC57 backone.
",s623="
Find our talk after the Regional Jamboree on the TEDx Youth Garza García website.

In accordance with TED’s mandate, we needed an idea worth spreading. We had a lot of ideas about our project, the iGEM competition and synthetic biology in general. The hard part was coming up with the right theme for our presentation; one that would resonate with the general public both within and outside Monterrey. One of the reasons we chose the project that we did is that it is something important to Monterrey and Mexico but that can also have implications around the globe – cancer research.
Beyond presenting the importance of getting involved in international university scientific competitions, we wanted to transmit to industry and government organizations, the importance of supporting synthetic biology university projects, which, if well supported, might define the next generation of innovations within the country.
",s475=" In order for TRAIL and Apoptin to exert their anticancer activity they must reach the extracellular matrix in the first place. This module uses the type I alpha-hemolysin secretion system of E. coli to achieve that feat.",s314="BB1: PrefixN // FNR Native promoter // FNR CDS // HIP-1 // RBS // suffixN",s269="In order for TRAIL and Apoptin to exert their anticancer activity they must reach the extracellular matrix in the first place. This module uses the type I alpha-hemolysin secretion system of E. coli to achieve that feat.

The mechanism of secretion is simple: In the cytosol, the substrate is recognized by means of a signal peptide and then translocated directly into the extracellular medium. The translocator consists of three proteins: HlyB, an ATP binding cassette; HlyD, a membrane fusion protein; and TolC, an outer membrane protein. Regarding the signal peptide, Apoptin and TRAIL were engineered to be fused C-terminally to the last 60 amino acids of HlyA (the natural substrate) since it has been shown that this C-terminal region of HlyA is both necessary and sufficient to direct secretion. While HlyB and HlyD are considered strain specific proteins, TolC is a component of multiple trans-membrane systems in many microorganisms, therefore we co-expressed only HlyB and HlyD.
",s460="FNR",s456="HlyA-Secretion",s630="TEDx",s574="Achievements",s278="The chicken anemia virus was discovered to be an avian pathogen that causes the eradication of thymocytes and erythroblastoid cells in chicks through the induction of apoptosis. It is a circular, single stranded DNA composed of approximately 2300 nucleotides. The CAV genome contains a 5’ non-transcribed region displaying promoter activity and within its genome there are three overlapping open reading frames, which produce a single strand of unspliced RNA. In all infected cells have been found three viral proteins (VP1, VP2 and VP3) translated from CAV RNA and the ORF2 encodes the 17 kDa protein VP3, this is also known as Apoptin, the protein that has been used as our therapeutic protein due to its apoptosis-inducing activity in various cancer cells lines. ",s461="His-TAT",s266="Enhanced Secretion
System",s563="Acknowledgment",s286="Therapeutic Proteins
Production",s722="General Methods",s721="Hi Tec Fest",s720="Workshops",s283="Apoptin is a small apoptosis-inducing protein from the chicken anemia virus composed of 121 amino acids. It is a proline-rich protein encoded by the VP3 gene of CAV, a member of the circovirus family.It appears to have innate tumor-specific, p53-independent, Bcl-2-enhanced proapoptotic activity and hence is of considerable interest for efficient targeting and specific elimination of cancer cells. The antitumor activity of apoptin appears to be linked to its ability to localize in the nucleus of infected cells, but not in those of primary or normal cells then more than 70 human tumor cell lines were shown to be susceptible.",s719="Cancer Manual",s718="Novel ownership and sharing approach",s717="Security and safety considerations",s324=" HIP-1 promoter",s672="Modelling",s131="Modular, synthetic biology approach for the development of a bacterial cancer therapy in Escherichia coli. ",s676="Hypoxia Promoters",s675="Therapeutic Proteins",s458="Apopptin",s653="Mexican Indigenous Communities Cancer-Self Examination Plan",s677="Secretion System",s557="Team
",s638="
The manual includes the basic information required for a self-examination breast cancer tumor. It had also been edited in .pdf format and a .mp3 format manual so the information could be highly managed by regional and national health institutions to which the formats are going to be provided.

This manual represents the first initiative to encourage public institutions to provide health related information within cancer prevention campaigns in the country.",s634="Those who have suffered from this disease and has exceeded the risk of relapse, hence the need to continue under observation for a period of five years, to avoid complications, which may be more aggressive than the first time, also because it generates resistance to conventional treatments.

During the last years, Mexican health institutions have made a great effort to educate the population with national and regional campaigns promoting cancer-self examination protocols, nevertheless, all the information given to the people is distributed and explained in Spanish, being this the official language of the country.

Nowadays, in Mexico there are more than 15 million indigenous people distributed in several communities across the country and approximately 5.4% of the Mexican population speaks an indigenous language. Therefore, there is an important lack of information distribution regarding health issues along the indigenous communities.

This year we wanted to set ourselves as pioneers in the field of making cancer related information accessible to these communities, so in order to promote breast cancer prevention, we decided to translate a complete breast self-examination manual to Otomí, one of the most spoken indigenous dialects in the country.

",s295="Chimeric promoter",s561="Members",s704="Colaboration Results",s620="This year our team had the opportunity to partner with TEDx Youth Garza García in our city Monterrey in November the 16th, and TEDx Coyoacán in Mexico City in December 17th.
This is a great opportunity to present to not only our city, but also a part of a larger, international event. We had a lot of fun thinking about synthetic biology and our project and the best way to share that in an inspirational and meaningful way.",s608="A wet lab workshop was organized for a group of 30 students from a local high school who came to the lab 4 sessions in which they learned the basics of synthetic biology, microbiology and molecular biology techniques such as mini plasmidic preparation and electrophoresis gel analysis. By the end of the workshop, the students were able to identify and grow their own skin bacteria and were also able to visualize and purify DNA.

Tec de Monterrey Santa Catarina Campus High School and Tec de Monterrey Cumbres Campus High School were visited to local high schools were also performed in order to present the project and the basic concepts of synthetic biology. We hope to make a difference in the lives of a few future biologists by introducing them to tools, techniques, and exciting applications from the cutting edge of synthetic biology.
",s298="The BioBrick produced contains:",s322="Based on the work of Mengesha,et al. (2006), a hypoxia-inducible promoter (HIP-1) was used to control the expression of green fluorescent protein (GFP). This promoter was originally obtained from a portion of Salmonella pepT promoter with the combination of two binding sites for the FNR regulon and a TATAAT sequence. Experimental work demonstrated that protein production was succesfull under acute and chronic hypoxia, but not under normoxia. To the correct operation of the promoter and all the anoxic methabolic pathways, there is one important gene: the fumarate and nitrate reductase (FNR). It is a transcriptional factor that inactives with the interaction of its sensory domain with oxigen molecules and it is present in Salmonella and E. coli.

For this project it was designed a construct for the expresion of GFP under hipoxic conditions to be used in BL21 E. coli strains. This constrsuct contains the FNR promoter and CDS native form E. coli, followed by a double terminator, the HIP-1 promoter dessinged by Mengesha, et al. (2006) and all the sequences required for the GFP expression. This expression cassete was cloned into a pUC57 plasmid.
",s307="According to Peakman, et al. (1990) the nirB gene is the required for the synthesis of the nitrite reductase apoprotein and cysG proteins required for the sirohaem synthesis.This protestetic group is fundamental in the anoxic methabolic pathways of E. coli and it is used by many enzymes to complete a six-electron reduction of sulfur and nitrogen. Knowing that these genes are specifically activated in E. coli during anoxic growth, in this project it was used the nirB promoter to make a cassette of expression to measure the effect of hypoxic environment over the expression of GFP.
The cassette is conformed by: the NirB promoter (BBa_K905000 sequence reported in iGEM) and all the sequences necessary to express GFP, all cloned in pUC57 vector and transformed in BL21 E. coli strain.
",s327="Tumor Specific
Induction",s656="As representatives of the iGEM competition and synthetic biology communities, our team aims to serve as a positive influence locally and globally. iGEM Tec Monterrey recognize significant broad impacts of our 2013 iGEM research project, both for potential therapeutic applications stemming from our novel technology and for the social impact that our Human Practice Project generated.

Furthermore, we aim to promote a wider understanding of the synthetic biology field within the local and global communities and have organized several outreach and educational programs to accomplish this goal.

Finally, we understand the importance of collaboration within the scientific community as a means of progress and have thus worked closely with fellow iGEM teams to foster positive connections and provide valuable assistance on their research projects.
",s310="Nir-B promoter",s566="Our University",s476="Internalization of Apoptin into the cytosol of human cells was confirmed using a Tat-GFP proof of concept approach.

The internalization module was characterized trough the use of Tat-GFP fusion protein purified by His-tag (HisPur, Thermo Scientific). Internalization assay was performed on coverslip grown human cells (NIH3, Caco-2) treated with 1 µg/mL, 5µg/mL and 10µg/mL Tat-GFP.",s462="Biobricks",s646="This year, iGEM Tec Monterrey is working on a novel bacterial therapy against cancer cells. Even when synthetic biology advances promise successful ways to treat cancer patients in a near future, our team knows that prevention will always be better than cure.

In Mexico, more than 3,500 women die every year for breast cancer, becoming the leading cause of deaths and the second most common tumor in this population. Moreover, one of the main problems of this type of malignancy in Mexico is the lack of early detection. Between 70 and 80 percent of patients go to to seek care in advanced stages of the disease.
",s591="Hi Tec Fest is the Tec de Monterrey University welcome event aimed to more than 2,000 freshmen students, its mission is to welcome the freshmen to college life at Monterrey Campus.

It also provides interaction with managers, teachers and strategic areas of the institution. It also seeks to encourage the love for his Alma Mater and engage in academic traditions and training co-curricular / integral life.

As part of the Hi Tec Fest itinerary, our team made a special presentation to show the project and to capture attention from more than 700 freshmen engineers students, the iGEM concept and the project description was very well accepted and we did got a lot of new students interested in the competition. This activity will be crucial to promote the continuity of iGEM teams in our University the following years.

",s528="Human Practice",s228="Conversación iniciada hoy
Luis Fernando Camarillo Guerrero
17:42
Luis Fernando Camarillo Guerrero
Future work
Validation by experts
An important aspect of every project is its validation by experts, although a project may seem perfect for its creators there could be hidden issues. Additionally, this validation can greatly increase the possibilities of funding your project.
With this in mind, we took on the task of looking for experts that could gave us feedback about our project.
Here’s the list of Drs. that we visited:
Dr. Luis Mario Villela – Escuela de Medicina y Ciencias de la Salud
Dr. Adolfo Isassi Chapa - San José Tec de Monterrey Hospital
Dr. Servando Cardona Huerta - San José Tec de Monterrey Hospital
Dr. Jorge Martínez - San José Tec de Monterrey Hospital
Dr. César González de León - Opción/ Muguerza
Dr. Augusto Rojas Martinez – Universidad Autónoma de Nuevo León
Dr. Alfonso Dueñas González – Instituto Nacional de Cancerología
We got invaluable feedback, particulary something that many Drs. pointed out was the sepsis issue. They thought it may be a better idea an intratumoral injection instead of an intravenous (i.v.) therapy, this with the goal of focalizing the inoculum and therefore reduce the interaction of the bacteria with the immune system
We also thought of some approaches that may be used in combination to tackle this problem.
Anti-LPS peptide
Lipopolysaccharide (LPS) is an ubiquitous cell surface component of E. coli and also one of the major toxins responsible for initiating sepsis (Lynn et al, 1992). Interestingly, it has been shown that the interaction of LPS with the immune system is mediated by a protein called LPS binding protein (LBP)(Jiang et al, 2000). Therefore, a way to reduce the risk of sepsis would be to block this interaction. It turns out that there are some molecules known to achieve that effect such as the Limulus anti-LPS factor (LALF), a small basic peptide found in hemocytes of the marine chelicerates Tachypleus tridentatus and Limulus polyphemus. An approach could be the administration of LALF during the therapy.
Oral therapy
The idea of administering bacteria orally in an effort to fight cancer already has been explored. It has been shown that Bifidobacterium breve fed to mice were detected specifically in tumors at levels similar to i.v. administration. (Cronin et al, 2010).
This was posible because some bacteria are able to translocate the gastrointestinal tract (GIT) including E. coli (Mahjoub-Messai et al, 2011).
Also, we thought that some additional elements must be included in our therapy:
Plasmid loss prevention
It’s well known that in absence of a selective pressure a plasmid can be lost if it doesn’t confer an advantageous trait. Inside the tumor where no antibiotic is found it’s critical to use an strategy to avoid this problem since plasmid loss will be reflected in loss of dosage of therapeutic proteins. A simple strategy would be an auxotrophic marker.
Horizontal gene transfer prevention
This issue already has been addressed on iGEM. A strategy could be the use of a toxin/anti-toxin system such as the Endolysin-Holin system proposed by the Imperical College of London.
References
Lynn, W.A., and D.T. Golenbock. (1992). Lipopolysaccharide antagonists.
Immunol. Today. 13(7):271–276.
Qingqi Jiang, Sachiko Akashi, Kensuke Miyake and Howard R. Petty. (2000). Cutting Edge: Lipopolysaccharide Induces Physical Proximity Between CD14 and Toll-Like Receptor 4 (TLR4) Prior to Nuclear Translocation of NF-κB B. J.. Immunol. 165:3541-3544.
Cronin M, Morrissey D, Rajendran S, El Mashad SM, van Sinderen D, O\'Sullivan GC, Tangney M. (2010). Orally administered bifidobacteria as vehicles for delivery of agents to systemic tumors. Mol Ther. 18(7):1397-407.
Mahjoub-Messai F, Bidet P, Caro V, Diancourt L, Biran V, Aujard Y, Bingen E, Bonacorsi S. (2011). Escherichia coli isolates causing bacteremia via gut translocation and urinary tract infection in young infants exhibit different virulence genotypes. J Infect Dis. 203(12):1844-9.",s459="HIP 1",s88="An important part of this Project is the localized expression of the therapeutic proteins. For more than sixty years it has been known that some bacteria grow preferably in hypoxic conditions, being E. coli one of these organisms. Tumors are known to be regions with low oxygen concentrations (hypoxic). With these factors in favor, three different hypoxia promoters were characterized.",s616="High School Outreach",s289="With these two BioBricks",s562="Advisors",s603="Our team also promoted the importance of synthetic biology in high school students interested in studying a science related major. Two different kinds of workshops were organized in order to show the students, basic concepts of synthetic biology and the importance of this new field in the development of future technology.",s599="Hi Tec Fest iGEM Presentation",s261="TRAIL",s152="By harnessing the inherent ability of facultative anaerobic bacteria to colonize and grow in tumoral environments, this project aims to prove the functionality of four different modules that would work together as a bacterial cancer therapy using Escherichia coli as chasis: Toxicity module, Secretion module, Localized induction module, and Internalization module.
The expression of tumor specific therapeutic proteins, Apoptin and TRAIL, conforms the toxicity module. For these proteins to have their effect they need to be located in the extracellular matrix, therefore we are developing a module with a secretion function using hemolysin secretory mechanism. The hypoxic microenvironment present in tumors can be used for the localized induction module of tumor specific proteins, using the promoters HIP and nirB. Finally, Apoptin needs mechanisms to enter tumor cells’ cytoplasm. Proteins with this requirement could reach the cytoplasm when coupled with the internalization module, resulting in a fusion with the TAT peptide.
",s292="According to the fundaments of the Hip-1 construct and the NirB promoter, it was designed a new cassette controlled by those parts over the expression of GFP:",s143="The bacterial system expresses two different proteins with the help of a hypoxia promoter. This experiment aims to prove the expression of the two toxins in E. coli (Apoptin and TRAIL). The expression of these tumor specific therapeutic proteins conforms the “Therapeutic proteins” module. More than 70 human tumor cell lines were shown to be susceptible to Apoptin, whereas Apoptin does not induce apoptosis in a variety of normal cells. TRAIL is a tumor specific agent under development as a novel anticancer therapeutic agent.",s257="Tumor Necrosis Factor-related Apoptosis Inducing Ligand or Apo2 known as TRAIL is a novel cytotoxic ligand belonging to the TNF superfamily, a transmembrana (type II) glycoprotein capable of inducing apoptosis in DR4, DR5, caspase-8 dependant manner.
TRAIL is a tumor specific agent under development as a novel anticancer therapeutic agent; it is a Type II transmembrana protein that binds to one of four receptors that have been identified in humans, TRAIL-R1/DR4, TRAIL-R2/KILLER/DR5, TRAIL-R3/DcR1/TRID and TRAIL-R4/DcR2/TRUNDD. DR4 and DR5 are pro-apoptotic receptors, which contain a cytoplasmic death domain and mediate apoptosis on binding to TRAIL.

Apoptosis is a process for the regulation of homeostasis and development and it can be induced by two different pathways. The first is the intrinsic pathway, the mitochondrial pathway that is induced by intracellular signals such as oncogene activation and the extrinsic pathway that is activated by cell surface death receptors. The activation of the death receptors by death ligand engagement induces the formation of a death-inducing signaling complex (DISC), once the DISC is formed, the caspase 8 is autoprocessed and activated by induced proximity. The TRAIL death ligand induces apoptosis via the extrinsic pathway.

However, certain TRAIL preparations against primary human hepotocytes have shown toxicity. Further studies suggested that this toxicity could be avoided by using native TRAIL but instead of tagged TRAIL. Therefore, it was necessary to use a Ni-NTA agarose column to obtain a soluble purified recombinant human TRAIL which doesn’t show toxicity. His6-tagged TRAIL form hexamers or nonomers TRAIL proteins that have the hability to kill the majority of the cell lines due to their toxicity.
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