Screening of different Mammalian cancer cell lines for Mycoplasma and Human Papillomavirus infection
Cancer cell lines
Cancer constitutes an enomerous burden in the society of developed and under developed countries. The reason for this could be aging of the population, growth as well as establishment of some risk factor such as smoking, overweight, physical inactivity etc. One of the leading causes for prevalence of cancer is HPV (Human Papillomavirus). Among known 120 types, HPV 16 and 18 are high risk HPV strains which causes cervical cancer whereas HPV 6 and 11 are low risk HPV strains which causes genital warts and rarely develop into cancer. HPV has been identified as a distinct causative agent in oropharyngeal cancer and has also been isolated in nasopharyngeal and oral cancer. Cervical cancer rank third in cancer incidence worldwide. The increasing rate of these diseases in developing countries is attributed to the early beginning of sexual activity such as high number of multiple partners, early age of first intercourse, frequent use of condoms and immunosuppression with HPV which is results in high risk of HPV infection.[3, 4]
Mammalian cancer cell lines are the most widely used models to study the biology of cancer and thus improve efficacy of this deadly state. Cell lines are preferred to be contamination free in order to carry out successful in-vitro experiments.
Contaminated cell lines may alter the normal physiology, biochemistry, functioning of normal cells and thereby compromising with overall reproducibility of experimental results. This lab is currently working on transcriptional profile of cervical cancer and HNSCC, so characterization of cell lines is one of the baseline studies for authenticity of experiments. Cancer cell line often gets contaminated with Mycoplasma so, screening of different Mammalian Cancer cell lines are obligatory.
Characterization of different mammalian cancer cell lines like Cervical cancer cell lines (C33a, SiHa, HeLa), Head and neck cancer cell lines (Oct-1, 93VU147T), Lung cancer cell line (A-549, H1299), Human prostate cancer cell line (DU145,PC3), Murine macrophage cancer cell line (RAW) and Human monocytic cell line (THP1) were studied using different PCR methods which include β-actin (DNA adequacy), PGMY09/11 (HPV detection), HPV 16 URR and HPV 18 E6 for cross contamination in order to perform HPV genotyping.
|CELL LINE||TISSUE & ORGAN||MORPHOLOGY||DISEASES||HPV STATUS|
|HeLa||Human, Cervix||Epithelial||Adenocarcinoma||16 positive|
|C33a||Human, Cervix||Epithelial||Adenocarcinoma, carcinoma||Negative|
|SiHa||Human, Cervix||Epithelial||Squamous cell carcinoma, Grade II||18 positive|
|OCT-1||Human, Oral||Epithelial||Oral cancer||Negative|
|93VU147T||Human, Head and neck||Squamous cell||Oral cancer||16 positive|
|UDSCC2||Human, Head and neck||Epithelial||Hypopharyngeal squamous cell carcinoma||16 positive|
|A549||Human, Lung||Epithelial||Lung adenocarcinoma||Negative|
|H1299||Human, Lung||Lymph node||Lung adenocarcinoma||Negative|
|DU145||Human, Prostrate||_____||Prostrate carcinoma, adenocarcinoma||Negative|
|PC3||Human, Prostrate||_____||Prostrate carcinoma, adenocarcinoma||Negative|
|THP1||Human Monocytic||Monocytic||Monocytic leukemia||Negative|
Human Papillomavirus (HPV)
Human Papillomavirus (HPV) are small double stranded, circular DNA viruses which live in flat, thin cells called epithelial cell. These are found on the skin’s surfaces, mouth, and throat and also in the surface of vagina, anus, vulva, cervix and head of the penis. About 100 of HPV are known which causes warts, some may enter the body during the time of sexual contact, but not all of them are responsible for serious health problem.
HPV16 and 18 are the high risk HPV strain which causes 70% of cervical cancer whereas HPV 6 and 11 are the low risk HPV strain which causes 90% genital warts which rarely develop into cancer. HPV comprises of a circular genome which is enclosed in a protein capsid made up of Late protein and Early protein. Late protein comprises of L1 and L2 type which is also an structural component of virus whereas Early protein may vary and are of E1 (helps in DNA replication), E2 (functions as viral RNA transcription), E4 (results in cytoskeleton reorganization), E5 (play role in membrane signaling protein), E6 and E7 (helps in oncogenic transformation )
Normal cell of our body is different from the one which is infected with HPV whether it is high risk or low risk strain and it may consist of different stages showing different phases of cancer.
In this report we will not only focus on HPV but will also study Mycoplasma contamination which effect the mammalian cancer cell line. Mycoplasma are the smallest prokaryotic organism that are bound by a triple layered membrane and can live independently without cell wall. The lack of cell wall is used to distinguish these micro-organism from ordinary bacteria and then include them in separate classes. It can be easily isolated from normal human and animal respiratory mucosa. In addition, mycoplasmas have the smallest amount of genetic information of any known cells, so their complexity is constrained by a limited genetic coding capacity. They are the small intracellular bacteria which get attaches to the cell membrane, inhibits their growth and can ultimately leads to cell death. That’s why its necessary for mammalian cancer cell line to be mycoplasma contamination free.
Out of the available techniques we have performed PCR to check the contamination in mammalian cancer cell line. The detection of Mycoplasma in Mammalian cancer cell line is mandatory because these bacteria are common contaminants and remain unrecognized for many years and effect the research result. PCR offers fast and simple technique to detect Mycoplasma, although it is very vulnerable to errors and can some time produce false negative as well as false positive results. Similarly, β-actin PCR is performed to check DNA adequacy in the cell where β-actin is also known as Housekeeping protein and is also used as a loading control. They can be used to normalize the level of protein detected by confirming that protein loading is the same across the gel. HPV consensus PCR was performed using primers PGMY09/11 designed to amplify a fragment of HPV L1 gene.
Objectives of the Research
The project is designed to study different types of contamination which includes bacterial, viral etc or any cross contamination between the cell lines. This study would be providing authenticated results essential for analysis of different cancerous cell lines.
Cell Lines Used
· Cervical cancer cell lines (C33a, SiHa, HeLa)
· Head and neck cancer cell lines (Oct-1, 93VU147T)
· Lung cancer cell line (A-549, H1299)
· Human prostate cancer cell line (DU145,PC3)
· Murine macrophage cancer cell line (RAW)
· Human monocytic cell line (THP1)
· DNA Lysis buffer
· Phenol Chloroform Isoamyl Alcohol
· Sodium acetate
· Absolute Ethanol
· 70% Ethanol
· 1XTE buffer
· Agarose powder
· ETBR (Ethidium Bromide)
This is an laboratory equipment used to stabilize temperature of water for a longer duration of time. It is also used to enable certain chemical reaction to occur at high temperature. The sample of different cancer cell lines were incubated at waterbath overnight for an temperature of 55°C.
Basically centrifuge works on the principal of Centrifugal force. The force from spinning that moves things away from the centre separates liquid that have different weights. A centrifuge were spin at 12,000rpm to facilitate separation of different phases of the extraction. After use it was cool and cleaned properly with tissues.
Incubator provides a controlled, contaminant-free environment for safe and reliable work with cells. It regulate and maintain the temperature, humidity which enables the pellet to dry for a duration of time.
Electrophoresis follow the mechanism of migration and separation. The –ve charge of its phosphate backbone move the DNA towards the +vely charge anode during Electrophoresis. 1XTE is used as an buffer which help the agarose gel to run.
It is also known as gel documentation system used for imaging and documentation of Nucleic acid and protein suspended with in polyacrylamide or agarose gel. Ethidium Bromide, Chemiluminescence, florescence are the detectors used to measure the nucleic acids and protein bands. Chemiluminescence are used during the time of western blotting whereas florescence are used for reading DNA or RNA extraction.
This instrument is designed for measuring nucleic acid concentration in sample volume of one microlitre. The ratio of absorbance at 260 nm versus 280 nm is commonly used to detect DNA contamination of protein solutions. A 260/280 ratio ̴ 1.8 is more often accepted as “pure” for DNA and a ratio ̴ 2.0 is accepected pure for RNA. The 260/230 ratio for “pure” nucleic acid are more higher than the values of 260/280 ratio. The considerate value for 260/230 ratio are in the range of 2.0-2.2.
The polymerase chain reaction is a sensitive technique by which a single DNA molecules can serve as a template for amplification. PCR reaction consists of different stages which include DNA denaturation for the separation of double stranded, primers association by hydrogen bands to the DNA target in a single chain and extension of the primers by synthesizing the complementary strand catalysed by DNA polymerase.
Concepts and Procedure
· Approximately 2-3 million cells are pelleted at 1500g for 5 minutes and store at 20°C or use fresh pellet. Stored pellet has to be thawed before resuspending.
· Resuspend the pellet in 0.2 ml of extraction solution/lysis buffer mix gently with pipette.
· Add Proteinase-K (stock concentration-5mg/ml) at working concentration of 50µg/ml to digest proteins.
· Incubate the tubes in a preheated water bath at 55°C for overnight.
· Add equal volume of PCIA in every tubes. Invert mix the tube by 10-20 times.
· Centrifuge at 12000rpm at 4°C for 7 minutes to separate the two liquid phase. Upper phase is aqueous phase containing DNA, lower phase is organic phenol phase.
· Take supernatant into a fresh tube and add equal volume of PCIA (50:25:24:1), and invert mix by 10-20 times.
· Repeat the following steps
· Take supernatant into a fresh tube and add equal volume of PCIA, invert mix the tube by 10-20 times.
· Again repeat the step.
· Take supernatant into a fresh tube and add 1/10th volume of 3M sodium acetate (pH 5.2)and twice the volume of chilled absolute ethanol to precipitate DNA.
· Centrifuge at 12,000rpm at 4°C for 20 minutes.
· Decant the supernatant and resuspend in 500µl 70%ethanol
· Centrifuge at 12,000rpm for 20 minutes
· Wash with 70% ethanol and dry the pellet.
· Dissolve the pellet in 1xTE buffer
· Check DNA concentration by agarose gel electrophoresis
RESULTS AND DISCUSSION
To check DNA concentration by Agarose Gel Electrophoresis
Agarose Gel Electrophoresis: 0.24g of agarose powder (Sigma) was dissolved in 30 ml 1X TAE. The gel was run at 74-90V until the dye line was approximately 75-80% of the way down the gel. Using GelDoc Amersham, the DNA fragments were visualized.
In both experiment except HeLa Bands were noted in every cell lines which indicates the presence of genomic DNA.
To check Bacterial Contamination through Mycoplasma PCR
PCR cycle (40 cycles) PCR mix × 10
|95°: 5 min||Master mix:125|
|55°: 30 sec||R. Primer: 05 ul|
|72°: 1 min||Template:20 ul|
|72°: 5min||NFW : 95 ul|
Agarose Gel Electrophoresis: The components were mixed properly and run on 2% agarose gel. 1g of agarose powder (Sigma) was dissolved in 50 ml 1X TAE buffer. The gel was run at 74-90V until the dye line was approximately 75-80% of the way down the gel. Using GelDoc Amersham, the DNA fragments were visualized.
|PCR Cycle (40 cycles): PCR mix × 9|
|95° : 5 Min||Master mix: 12.5ul|
|94°: 30 sec||F. Primer: 0.5ul|
|55°: 30 sec||R. Primer: 0.5ul|
|72°: 1 min||Template DNA: 2ul|
|72°: 5min||NFW : 9.5ul|
Two experiments were performed. First one with untreated cells which shows the presence of bands indicating mycoplasmic contamination. The same cell lines were repeated second time just after the treatment and they shows absence of bands indicating contamination free cell lines.
To check DNA Adequacy with the help of β-actin PCR
PCR Cycle (40 cycle)- PCR mix × 9
|95°: 5 min||IQ SYBR Green Supermix: 12.5 ul|
|95°: 30 sec||F. Primer: 0.5 ul|
|56°: 45 sec||R. Primer: 0.5 ul|
|72°: 60 sec||Template: 2ul|
|72°: 5min||ddH2O : 9.5ul|
Each cell lines shows the presence of band at 432bp leaving –ve control which indicated in β-actin PCR, DNA adequacy was checked.
To detect the Presence of HPV with the help of PGMY09/11 PCR
PCR Cycle (45 cycle)
|95°: 5 Min||Mastermix: 12.5 ul|
|95°: 30 sec||F. Primer: 0.88ul (88 nM)|
|55°: 90 sec||R. Primer: 0.88ul (88 nM)|
|72°: 120 sec||HLA: 0.44nM (0.44ul)|
|72°: 5min||Template: 2 ul|
|NFW: 8.74 ul|
The components were mixed properly and run on 2% agarose gel. Using GelDoc Amersham, the DNA fragments were visualized.
Leaving other cell line a smear is formed only in SiHa, HeLa & RAW indicating L1 gene of 450bp and HLA gene of 220bp.
- Fang, J., H. Zhang and S. Jin (2014). "Epigenetics and cervical cancer: from pathogenesis to therapy." Tumour Biol35(6): 5083-5093.
- Kaarthigeyan, K. (2012). "Cervical cancer in India and HPV vaccination." Indian J Med Paediatr Oncol33(1): 7-12.
- Maxwell, J. H., J. R. Grandis and R. L. Ferris (2016). "HPV-Associated Head and Neck Cancer: Unique Features of Epidemiology and Clinical Management." Annu Rev Med67: 91-101.
- Shimizu, T. (2015). "[Pathogenic factors of mycoplasma]." Nihon Saikingaku Zasshi70(4): 369-374.
- Tsikouras, P., S. Zervoudis, B. Manav, E. Tomara, G. Iatrakis, C. Romanidis, A. Bothou and G. Galazios (2016). "Cervical cancer: screening, diagnosis and staging." J BUON21(2): 320-325.
- Human papilloma virus statistics from https://www.columbiamissourian.com/visuals/graphics/hpv-statistics/image_f322770c-c01c-11e7-9ad1-6711036b0f37.html
- Human_Papilloma_Virus from http://microbewiki.kenyon.edu/index.php
First, I would like to thank IASc-INSA-NASI Summer Fellowship programme for providing me this opportunity to be able to conduct scientific research in the form of a short summer project.
I would like to thank Prof. Alok Chandra Bharti, my guide, for mentoring me.
I would like to express my gratitude to Ms. Nikita Agrawal and Ms. Joni Yadav for their support and guidance.
I would also like to thank my labmembers Mr.Mohit Jadli, Mr. Tejveer Singh,Ms. Anjali Bhat, Mr. Kulbushan Thakur, Mr. Arun Chhokar, Ms. Rakhi and Ms. Ujala Singh for their constant encouragement.