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Summer Research Fellowship Programme of India's Science Academies

Screening of different Mammalian cancer cell lines for Mycoplasma and Human Papillomavirus infection

Smriti Sneh Verma

Department of Bioscience and Biotechnology, Banasthali Vidyapith, Newai, Tonk 304022

Prof. Alok Chandra Bharti

Department of Zoology, University of Delhi, Delhi 110007

Abstract

Cancer is one of the major causes of death around the world in which cells grow out of control, divide and invade other tissues. The cancer cells change their nature because of mutation and hence their daughter cells also become cancerous. There are different types of cancer, out of which Breast cancer, Head and neck cancer and Lung cancer are the most prevalent types. Head and neck cancers comprises of the oropharynx and hypopharynx than the oral cavity or larynx. The major cause for HNSCC is alcohol consumption & these days prevelance of HPV in HNSCC (Head and neck squamous cell carcinoma) is being reported whereas cervical cancer, mainly caused by Human Papillomavirus infection, is the leading cancer in Indian women and the second most common cancer in women worldwide. Since most of the cases of cervical cancer have been found associated with HPV, additional genetic and epigenetic changes are required for disease progression. A unique aspect of human papillomavirus involves dependence on the differentiation status of the host epithelial cell to complete the viral life cycle. A small group of these viruses are the etiologic agents of several types of human cancers, including oral and anogenital tract carcinomas. Mammalian cancer derived 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 and in-vivo experiments. Contaminated cell lines may alter the normal physiology, biochemistry, functioning of normal cells and thereby compromising with overall reproducibility of experimental results. This contamination could vary and may be of different type such as fungi, bacteria or mycoplasma contamination. Mycoplasma is the smallest micro-organism which lack cell wall and savor a free lifestyle. Since the mammalian cells get easily contaminated with mycoplasma and HPV, to detect this contamination in mammalian cancer cell line different PCRs (Polymerase chain reaction) were performed.

Keywords: cancer biology, Mammalian cancer cell lines, Mycoplasma, Human papillomavirus, cervical cancer cell, HNSCC

INTRODUCTION

Background

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]

ssv_1.jpg
    Percentages of new cancer cases and cancer deaths in worldwide in 2018

    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.

    Characterization of Mammalian cancer cell lines
    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
    LNcAP Human, Prostrate ______ Adenocarcinoma 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.

    HPV body 1.jpg.png
      HPV infection

      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 )

      HPV protein.png
        HPV protein cycle

        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.

        HPV image2.jpg
          Fig.2.Different stages of HPV[6]

          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.

          mycoplasma changed.png
            Structure of Mycoplasma

            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.

            METHODOLOGY

            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) 

            Chemicals Used

            ·         DNA Lysis buffer

            ·         Proteinase-K

            ·         Phenol Chloroform Isoamyl Alcohol

            ·         Sodium acetate

            ·         Absolute Ethanol

            ·         70% Ethanol

            ·         1XTE buffer

            ·         Agarose powder

            ·         ETBR (Ethidium Bromide)

            Instruments Used

            Waterbath

            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.

            4°Centrifuge

            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

            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

            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.

            GelDoc Amersham

            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.

            Nanodrop

            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.

            PCR

            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

            First experiment

            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.

            gd1.png
              Geldoc view of gel

              Nanodrop reading:

              Cell lines ng/µl 260/280 260/230
              C33a 638.3 1.97 2.25
              SiHa 707.2 1.96 2.21
              HeLa 2459.2 2.01 2.09
              OCT-1 215.2 1.82 1.30
              VU147T 602.9 1.96 2.27
              A549 843.8 1.97 2.08
              RAW 650.0 1.95 2.21

              Second Experiment

              gd2.png
                Geldoc view of gel

                Nanodrop Reading:

                Cell lines ng/µl 260/280 260/230
                OCT-1 68.6 1.85 1.48
                UDSCC2 102.8 1.85 1.23
                HeLa 161.7 1.93 1.35
                A549 80.4 1.80 1.65
                H1299 169.9 1.92 1.46
                DU145 87.1 1.87 0.67
                RAW 184.8 1.90 1.76
                THP1 139.9 1.83 1.91

                Discussion

                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

                First experiment

                Reaction conditions:

                PCR cycle (40 cycles) PCR mix × 10

                95°: 5 min Master mix:125
                94°:30 sec F.Primer:05µl
                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.

                gd3.png
                  geldoc view of gel

                  Second Experiment

                  Reaction conditions:

                  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
                  gd4.png
                    Mycoplasma PCR geldoc view

                    Discussion

                    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

                    Reaction conditions:

                    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
                    gd5.png
                      Geldoc view of gel

                      Discussion:

                      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

                      Reaction conditions:

                      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.

                      gd6.png
                        .Geldoc view of gel

                        Discussion:

                        Leaving other cell line a smear is formed only in SiHa, HeLa & RAW indicating L1 gene of 450bp and HLA gene of 220bp.

                        REFERENCES

                        • 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​

                        ACKNOWLEDGEMENTS

                        I would like to thank everyone who is involved in this project.

                        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.

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