Accessibility / Report Error

Alternatives for obtaining a continuous cell line from Apis mellifera

Alternativas para a obtenção de uma linhagem celular contínua de abelhas Apis mellifera

ABSTRACT:

In worldwide there are reports of a significant decrease in colonies of the species Apis mellifera, caused by several factors, including viral infections. In order to study and diagnose illnesses caused by viruses, in vitro cell culture is used as a valuable tool. Yet, there are still no immortalized cell lines of honey bee Apis mellifera. Primary cell cultures are promising for this purpose and can supply the lack of continuous strains, but their establishment is difficult and laborious, which often makes them unfeasible for many research centers. Through the use of cell immortalization techniques, it is possible to develop continuous cell lines and thus benefit, in different ways, research related to different species of bees. The choice of technique is challenging, since in addition to the ability to remain viable for countless passages, cells must keep the genotype and phenotype similar or identical to the original tissue. This review intends to present methodologies that can be used to immortalize Apis mellifera cells, aiming to establish a cell line. The genotypic and phenotypic implications of each technique are evaluated, and the purpose of the cell line to be developed.

Key words:
immortalization; cell culture; cell line; honeybee

RESUMO:

Ao redor do mundo há relatos da diminuição significativa de colônias da espécie Apis mellifera, causada por diversos fatores, incluindo infecções virais. Para estudo e diagnóstico de enfermidades causadas por vírus utiliza-se, como uma ferramenta valiosa, o cultivo celular in vitro. Contudo, ainda não existem linhagens celulares imortalizadas de abelhas Apis mellifera. Os cultivos celulares primários são promissores para este fim e podem suprir a falta de linhagens contínuas, porém seu estabelecimento é difícil e laborioso o que, muitas vezes, os torna inviáveis para muitos centros de pesquisa. Através do uso de técnicas de imortalização celular é possível desenvolver linhagens contínuas de células e assim beneficiar, de diversas formas, as pesquisas relacionadas às diferentes espécies de abelhas. A escolha da técnica é desafiadora, visto que, além da capacidade de permanecer viável por inúmeras passagens, as células devem manter o genótipo e fenótipo semelhante ou idêntico ao tecido original. O objetivo deste trabalho é apresentar metodologias que podem ser utilizadas para imortalização de células de Apis mellifera, visando o estabelecimento de uma linhagem celular. São avaliadas as implicações genotípicas e fenotípicas de cada técnica, e a finalidade da linhagem celular a ser desenvolvida.

Palavras-chave:
imortalização; cultivos celulares; linhagem celular; abelhas

INTRODUCTION:

The combination of factors, such as environmental stress and infections by various pathogens, are causing a drastic reduction in bee populations across the planet (PIRES et al., 2016PIRES, C. S. S. et al. Weakness and collapse of bee colonies in Brazil: are there cases of CCD?. Pesquisa Agropecuária Brasileira, v.51, n.5, p.422-442, 2016. Available from: <Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-204X2016000500422 >. Accessed: Nov. 04, 2019. doi: 10.1590/S0100-204X2016000500003.
http://www.scielo.br/scielo.php?script=s...
). Viral infections pose a significant threat to beekeeping (GISDER & GENERSCH, 2017GISDER, S.; GENERSCH, E. Viruses of commercialized insect pollinators. Journal of Invertebrate Pathology, v.147, p.51-59, 2017. Available from: <Available from: https://www.sciencedirect.com/science/article/abs/pii/S0022201116300957 >. Accessed: Jul. 12, 2019. doi: 10.1016/j.jip.2016.07.010.
https://www.sciencedirect.com/science/ar...
). Viruses can persist naturally in colonies, using a variety of transmission and replication pathways, and often may not cause visible symptoms. However, the presence of these microorganisms, associated with the accumulation of environmental factors, can trigger the death of an entire colony (VANENGELSDORP et al., 2017VANENGELSDORP, D. et al. Colony Collapse Disorder (CCD) and bee age impact honey bee pathophysiology. PLoS One, v.12, n.7, e0179535, 2017. Available from: <journals.plos.org/plosone/article?id=10.1371/journal.pone.0179535>. Accessed: Oct. 11, 2019. doi: 10.1371/journal.pone.0179535.
https://doi.org/10.1371/journal.pone.017...
). An increasing number of viruses have been identified in Apis mellifera, many of them being associated with the disappearance of these bees (BEAUREPAIRE et al., 2020BEAUREPAIRE, A. et al. Diversity and global distribution of viruses of the western honey bee, Apis melífera. Insects, v.11, n.4, p.1-25, 2020. Available from: <Available from: https://www.mdpi.com/2075-4450/11/4/239 >. Accessed: Sep. 14, 2020. doi: 10.3390/insects11040239.
https://www.mdpi.com/2075-4450/11/4/239...
).

Viruses require the help of cellular machinery to replicate and produce new particles (VIRGIN et al., 2009VIRGIN, H. W. et al. Redefining chronic viral infection. Cell, v.138, n.1, p.30-50, 2009. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S0092867409007831 >. Accessed: Nov. 17, 2020. doi: 10.1016/j.cell.2009.06.036.
https://www.sciencedirect.com/science/ar...
), which makes cell lines a valuable tool for studying the interaction of these microorganisms with cells, gene expression, DNA and RNA replication, among others (GENERSCH et al., 2013GENERSCH, E. et al. Standard methods for cell cultures in Apis mellifera research. Journal of Apicultural Research, v.52, n.1, p.1-8, 2013. Available from: < Available from: https://www.tandfonline.com/doi/abs/10.3896/IBRA.1.52.1.02 >. Accessed: Jun. 24, 2020. doi: 10.3896/IBRA.1.52.1.02.
https://www.tandfonline.com/doi/abs/10.3...
). A cell line can be obtained from primary cultures, which generally give rise to finite lines, with few passages and low proliferation. However, neoplastic or immortalized tissues can give rise to continuous cell lines, with a high rate of cell proliferation and an indefinite number of passages (CASTILHO et al., 2008CASTILHO, L., et al. Animal cell technology: from biopharmaceuticals to gene therapy. New York : Garland Science, 2008.). Yet, the lack of immortalized cell cultures of Apis mellifera has been a limiting factor for research in this species (CARRILLO-TRIP et al., 2016) This review aims to discuss viable alternatives for obtaining a continuous cell line of Apis mellifera bees, through techniques of cell immortalization.

Cell culture

Since the development of the first invertebrate cell lines (GRACE, 1962GRACE, T. D. Establishment of four strains of cells from insect tissues grown in vitro. Nature, v.195, p.788-789, 1962. Available from: <cabdirect.org/cabdirect/abstract/19632900172>. Accessed: Oct. 22, 2019. doi: 10.1038/195788a0.
https://doi.org/10.1038/195788a0....
), more than 1100 insect cell lines have already been registered, according to the Cellossaurus® database (ExPASy - Swiss Institute of Bioinformatics -SIB, 2020). Among them, only two of Apis mellifera, AmE-711 (GOBLIRSCH et al., 2013GOBLIRSCH, M. A cell line resource derived from honey bee (Apis mellifera) embryonic tissues. PLoS One, v.8, n.7, 2013. Available from: <journals.plos.org/plosone/article?id=10.1371/journal.pone.0069831>. Accessed: Nov. 15, 2019. doi: 10.1371/journal.pone.0069831.
https://doi.org/10.1371/journal.pone.006...
) and MYN9 (KITAGISHI et al., 2011KITAGISHI, Y. et al. Long-term cultivation of in vitro Apis mellifera cells by gene transfer of human c-myc proto-oncogene. In Vitro Cellular & Developmental Biology-Animal, v.47, n.7, p.451-453, 2011. Available from: <pubmed.ncbi.nlm.nih.gov/21688083/>. Accessed: Sep. 21, 2019. doi: 10.1007/s11626-011-9431-6.
https://doi.org/10.1007/s11626-011-9431-...
), considering all insect species, are described. The AmE-711 is originated from honeybee eggs and is a strain that has not undergone an immortalization process, and presents characteristics of a finite lineage, such as being ,is limited to a few passages, low cell proliferation and is the one which the scientific society does not have commercial access. This cell line proved difficult to maintain and crashed in 2015 but have been recovered and adapted to a commercially medium (CARRILLO-TRIPP et al., 2016CARRILLO-TRIPP, J. et al. In vivo and in vitro infection dynamics of honeybee viruses. Scientific reports, v.6, p.22265, 2016. Available from: <Available from: https://www.nature.com/articles/srep22265 >. Accessed: Nov. 01, 2020. doi: 10.1038/srep22265.
https://www.nature.com/articles/srep2226...
; BEAUREPAIRE et al., 2020BEAUREPAIRE, A. et al. Diversity and global distribution of viruses of the western honey bee, Apis melífera. Insects, v.11, n.4, p.1-25, 2020. Available from: <Available from: https://www.mdpi.com/2075-4450/11/4/239 >. Accessed: Sep. 14, 2020. doi: 10.3390/insects11040239.
https://www.mdpi.com/2075-4450/11/4/239...
; GUO et al., 2020GUO, Y. et al. Cell lines for honey bee virus research. Viruses, v.12, n.2, p.236, 2020. Available from: < Available from: https://www.mdpi.com/1999-4915/12/2/236 >. Accessed: Oct. 03, 2020. doi: 10.3390/v12020236.
https://www.mdpi.com/1999-4915/12/2/236...
). The MYN9 is originated from honeybee larvae and is the only report of cultivation of this species that was induced to immortalization. The immortalization was performed using the introduction of human c-myc proto-oncogene by conventional lipofection technique, and subculture at eight months until the end of project (KITAGISHI et al., 2011KITAGISHI, Y. et al. Long-term cultivation of in vitro Apis mellifera cells by gene transfer of human c-myc proto-oncogene. In Vitro Cellular & Developmental Biology-Animal, v.47, n.7, p.451-453, 2011. Available from: <pubmed.ncbi.nlm.nih.gov/21688083/>. Accessed: Sep. 21, 2019. doi: 10.1007/s11626-011-9431-6.
https://doi.org/10.1007/s11626-011-9431-...
). However, this strain is not available in any cell bank, and there are no reports of its use.

As a way to overcome this situation of the unavailability of Apis mellifera cell lines, studies with viruses, currently carried out, use primary or subcultivated cultures from different tissues and at different stages of development of this species (GENERSCH et al., 2013GENERSCH, E. et al. Standard methods for cell cultures in Apis mellifera research. Journal of Apicultural Research, v.52, n.1, p.1-8, 2013. Available from: < Available from: https://www.tandfonline.com/doi/abs/10.3896/IBRA.1.52.1.02 >. Accessed: Jun. 24, 2020. doi: 10.3896/IBRA.1.52.1.02.
https://www.tandfonline.com/doi/abs/10.3...
; BEAUREPAIRE et al., 2020BEAUREPAIRE, A. et al. Diversity and global distribution of viruses of the western honey bee, Apis melífera. Insects, v.11, n.4, p.1-25, 2020. Available from: <Available from: https://www.mdpi.com/2075-4450/11/4/239 >. Accessed: Sep. 14, 2020. doi: 10.3390/insects11040239.
https://www.mdpi.com/2075-4450/11/4/239...
). So far, several primary cultures with 6 to 135 days have been reported, originating from eggs, pupae, intestines, nervous tissue, among others (POPPINGA et al., 2012POPPINGA, Lena et al. Identification and functional analysis of the S-layer protein SplA of Paenibacillus larvae, the causative agent of American Foulbrood of honey bees. PLoS Pathog, v.8, n.5, p.e1002716, 2012. Available from: <Available from: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1002716 >. Accessed: Nov. 01, 2020. doi: 10.1371/journal.ppat.1002716.
https://journals.plos.org/plospathogens/...
; JU & GHIL, 2015JU, H.; GHIL, S. Primary cell culture method for the honeybee Apis mellifera. In Vitro Cellular & Developmental Biology-Animal, v.51, n.9, p.890-893, 2015. Available from: <link.springer.com/article/10.1007/s11626-015-9924-9>. Accessed: Oct. 28, 2020. doi: 10.1007/s11626-015-9924-9.
https://doi.org/10.1007/s11626-015-9924-...
; GUO et al., 2020GUO, Y. et al. Cell lines for honey bee virus research. Viruses, v.12, n.2, p.236, 2020. Available from: < Available from: https://www.mdpi.com/1999-4915/12/2/236 >. Accessed: Oct. 03, 2020. doi: 10.3390/v12020236.
https://www.mdpi.com/1999-4915/12/2/236...
).

The Laboratory of Virology and Immunology, at the Veterinary Faculty of Universidade Federal de Pelotas - UFPel - has been developing studies for the stabilization of primary cultures and the immortalization of a cell line of Apis mellifera. So far, our research group has accumulated promising results, with the development of more than 60 primary cultures originating from eggs and bee larvae, which have remained viable for more than 4 months and with several passages. The main problems observed, such as contamination, culture media and cultivation patterns, have already been overcome. Our group also successfully managed primary cultures maintaining a high rate of proliferation even after eight months of freezing. This fact is extremely relevant, as it allows continuous work in the laboratory even in winter, when there is no laying by the queen, which would make it impossible to make primary cultures from eggs. The next step is the elaboration of a continuous lineage of this species, through cellular immortalization.

Alternatives to cell immortalization

In general, cell immortalization occurs when the cell loses the pathways of verification of the cell cycle, responsible for senescence and apoptosis (WRIGHT & SHAY, 1992WRIGHT, W. E.; SHAY, J. W. The two-stage mechanism controlling cellular senescence and immortalization. Experimental Gerontology, v.27, p.383-389, 1992. Available from: <europepmc.org/abstract/med/1333985>. Accessed: Nov. 06, 2019. doi: 10.1016/0531-5565(92)90069-c.
https://doi.org/10.1016/0531-5565(92)900...
; MAQSOOD et al., 2013MAQSOOD, M. I. et al. Immortality of cell lines: challenges and advantages of establishment. Cell Biology International, v.37, n.10, p.1038-1045, 2013. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/23723166/ >. Accessed: Nov. 01, 2020. doi: 10.1002/cbin.10137.
https://pubmed.ncbi.nlm.nih.gov/23723166...
). Among some genes related to these processes, p53, p16 and pRb stand out (BARNES et al., 2019BARNES, P. J. et al. Cellular senescence as a mechanism and target in chronic lung diseases. American journal of respiratory and critical care medicine, v.200, n.5, p.556-564, 2019. Available from: < Available from: https://www.atsjournals.org/doi/full/10.1164/rccm.201810-1975TR >. Accessed: May, 17, 2020. doi: 10.1164/rccm.201810-1975TR.
https://www.atsjournals.org/doi/full/10....
). The establishment of a cell line can be achieved through the adoption of several immortalization techniques or protocols, including the use of radiation, carcinogenic agents, chemicals, viruses and recombinant DNA vectors that express oncogenes (STACEY, 2006STACEY, G. Primary Cell Cultures and Immortal Cell Lines. Encyclopedia of Life Sciences, p.1-5, 2006. Available from: <Available from: https://onlinelibrary.wiley.com/doi/10.1038/npg.els.0002564 >. Accessed: Nov. 04, 2019. doi: 10.1038/npg.els.0002564.
https://onlinelibrary.wiley.com/doi/10.1...
). Cell immortalization is seen as a challenge for scientific communities and, in the case of insect cells, this challenge becomes even greater due to the lack of genetic methods for derivation of new cell lines (LI et al., 2012LI, X. et al. A new insect cell line from pupal ovary of Spodoptera exigua established by stimulation with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). In Vitro Cellular & Developmental Biology-Animal, v.48, n.5, p.271-275, 2012. Available from: <link.springer.com/article/10.1007/s11626-012-9511-2>. Accessed: Jul. 22, 2020. doi: 10.1007/s11626-012-9511-2.
https://doi.org/10.1007/s11626-012-9511-...
; MAQSOOD et al., 2013MAQSOOD, M. I. et al. Immortality of cell lines: challenges and advantages of establishment. Cell Biology International, v.37, n.10, p.1038-1045, 2013. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/23723166/ >. Accessed: Nov. 01, 2020. doi: 10.1002/cbin.10137.
https://pubmed.ncbi.nlm.nih.gov/23723166...
). Some examples of immortalized cell lines are shown in table 1.

Table 1
Agents of immortalization, targets, and cell lines.

myc Proto-oncogenes

The myc gene group has great importance in the cell cycle, as the increase in its expression directly affects the transcription of genes responsible for cell proliferation, transformation, differentiation, metabolism, genomic maintenance and adhesion, but also apoptosis (DANG et al., 2006DANG, C. V. et al. The c-Myc target gene network. Seminars in Cancer Biology, v.16, n.4, p.253-264, 2006. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16904903 >. Accessed: Nov. 05, 2019. doi: 10.1016/j.semcancer.2006.07.014.
https://www.ncbi.nlm.nih.gov/pubmed/1690...
; GARCÍA-GUTIÉRREZ et al., 2019GARCÍA-GUTIÉRREZ, L. et al. MYC oncogene contributions to release of cell cycle brakes. Genes, v.10, n.3, p.244, 2019. Available from: < Available from: https://www.mdpi.com/2073-4425/10/3/244 >. Accessed: Jul. 23, 2020. doi: 10.3390/genes10030244.
https://www.mdpi.com/2073-4425/10/3/244...
). The Induction of an increase in intracellular myc levels develop a mutagenic effect and also cellular stress. The mutagenic and cell stress environment enhance the rate in intracellular oxygen free radical and can promote the p53 protein phosphorylation, which is known as the main gene responsible of cellular apoptosis and senescence (VAFA et al., 2002VAFA, O. et al. c-Myc can induce DNA damage, increase reactive oxygen species, and mitigate p53 function. A mechanism for oncogene-induced genetic instability. Molecular Cell, v.9, n.5, p.1031-1044, 2002. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/12049739 >. Accessed: Nov. 12, 2019. doi: 10.1016/s1097-2765(02)00520-8.
https://www.ncbi.nlm.nih.gov/pubmed/1204...
; WOLPAW & DANG, 2018WOLPAW, A. J.; DANG, C. V. MYC-induced metabolic stress and tumorigenesis. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer, v.1870, n.1, p.43-50, 2018. Available from: <pubmed.ncbi.nlm.nih.gov/29791870/>. Accessed: Oct. 12, 2020. doi: 10.1016/j.bbcan.2018.05.003.
https://doi.org/10.1016/j.bbcan.2018.05....
). The set of changes such as inhibition of apoptosis and cell transformation can promote cell proliferation even in non-nutritious culture media and in the absence of growth factors (EILERS & EISENMAN, 2008EILERS, M.; EISENMAN, R. N. Myc’s broad reach. Genes & Development, v.22, p.2755-2766, 2008. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/18923074 >. Acessed: Nov. 08, 2019. doi: 10.1101/gad.1712408.
https://www.ncbi.nlm.nih.gov/pubmed/1892...
).

The importance of myc oncogenes in malignancy and maintenance of transformed cells is clearly reported in the literature (KABILOVA et al., 2006KABILOVA, T. O. et al. Inhibition of human carcinoma and neuroblastoma cell proliferation by anti-c-myc siRNA. Oligonucleotides, v.16, n.1, p.15-25, 2006. Available from: < Available from: https://www.liebertpub.com/doi/abs/10.1089/oli.2006.16.15 >. Accessed: Sep. 22, 2020. doi: 10.1089/oli.2006.16.15.
https://www.liebertpub.com/doi/abs/10.10...
; PINTO et al., 2019PINTO, L. C. et al. Mebendazole induces apoptosis via C-MYC inactivation in malignant ascites cell line (AGP01). Toxicology in Vitro, v.60, p.305-312, 2019. Available from: <pubmed.ncbi.nlm.nih.gov/31207347/>. Accessed: Jun, 18, 2020. doi: 10.1016/j.tiv.2019.06.010.
https://doi.org/10.1016/j.tiv.2019.06.01...
). Inhibition of c-myc expression through siRNA in squamous cell carcinoma (SK-N-MC) and neuroblastoma (KB-3-1) cell lines led to loss of cell proliferation (KABILOVA et al., 2006). Mebendazole, which showed anti-cancer activity, was used in gastric cancer cell lines (AGP01), and induced apoptosis due to inhibiting the expression of c-myc (PINTO et al., 2019).

The only transformation of bee cells in vitro, described in the literature, was performed by Kitagish et al. (2011) with the insertion of six human proto-oncogenes in bee cells, using the conventional lipofection technique. Among them, pcDNA3 c-myc resulted in a cell line, named MYN9, with more than 100 passages. However, there is no other report of this lineage in the literature or deposit in a cell bank. It is believed that the use of c-myc genes such as pcDNA3 c-myc presents itself as a viable option for the development of a cell line, authenticated and available to other researchers.

Ras proto-oncogene

Ras proteins belong to the superfamily of Ras genes (WENNERBERG et al., 2005WENNERBERG, K. et al. The Ras superfamily at a glance. Journal of cell science, v.118, n.5, p.843-846, 2005. Available from: <Available from: https://jcs.biologists.org/content/118/5/843 >. Accessed: Jul. 11, 2020. doi: 10.1242/jcs.01660.
https://jcs.biologists.org/content/118/5...
). These proteins activate signaling networks that control cell proliferation, growth, and survival (WHITE et al., 1995WHITE, M. A. et al. Multiple ras functions can contribute to mammalian cell transformation. Cell, v.80, n.4, p.533-541, 1995. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/0092867495905073 >. Accessed: Nov. 07, 2019. doi: 10.1016/0092-8674(95)90507-3.
https://www.sciencedirect.com/science/ar...
; SIMANSHU et al., 2017SIMANSHU, D. K. et al. RAS proteins and their regulators in human disease. Cell, v.170, n.1, p.17-33, 2017. Available from: < Available from: https://www.sciencedirect.com/science/article/pii/S0092867417306505 >. Accessed: Jun. 26, 2020. doi: 10.1016/j.cell.2017.06.009.
https://www.sciencedirect.com/science/ar...
). Ras mutations make their proteins constantly expressed, which is widely seen in neoplasms (PRIOR et al., 2012PRIOR, I.A. et al. A comprehensive survey of Ras mutations in cancer. Cancer research, v.72, n.10, p.2457-2467, 2012. Available from: <cancerres.aacrjournals.org/content/72/10/2457>. Accessed: Jun. 20, 2020. doi: 10.1158/0008-5472.can-11-2612.
https://doi.org/10.1158/0008-5472.can-11...
). About 19% of neoplasms in humans have some mutant isoform of the Ras genes continuously expressed (PRIOR et al., 2020PRIOR, I. A. et al. The frequency of Ras mutations in cancer. Cancer Research, v.80, n.14, 2020. Available from: <cancerres.aacrjournals.org/content/80/14/2969>. Accessed: Oct. 17, 2020. doi: 10.1158/0008-5472.CAN-19-3682.
https://doi.org/10.1158/0008-5472.CAN-19...
). This continuous activation of Ras results in over-stimulation of it signaling pathways and drives the growth and survival of neoplastic cells (HOLDERFIELD, 2018HOLDERFIELD, M. Efforts to develop KRAS inhibitors. Cold Spring Harbor perspectives in medicine, v.8, n.7, 2018. Available from: <perspectivesinmedicine.cshlp.org/content/8/7/a031864>. Accessed: Sep. 14, 2020. doi: 10.1101/cshperspect.a031864.
https://doi.org/perspectivesinmedicine.c...
).

The activation of the Ras gene was able to cause an increase in cell proliferation of Drosophila sp (KARIM & RUBIN, 1998KARIM, F. D.; RUBIN, G. M. Ectopic expression of activated Ras1 induces hyperplastic growth and increased cell death in Drosophila imaginal tissues. Development, v.125, n.1, p.1-9, 1998. Available from: <dev.biologists.org/content/125/1/1>. Accessed: May, 18, 2020.; BRUMBY & RICHARDSON, 2003BRUMBY, A. M.; RICHARDSON, H. E. Scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila. The EMBO journal, v.22, n.21, p.5769-5779, 2003. Available from: < Available from: https://www.embopress.org/doi/full/10.1093/emboj/cdg548 >. Accessed: Jun. 22, 2020. doi: 10.1093/emboj/cdg548.
https://www.embopress.org/doi/full/10.10...
). The mutant Ras V12 gene had its efficiency proven in the immortalization of Drosophila cells, reducing the time for the confluence of the cells and allowing the cells to be cultured by several passages. Using this oncogene, several Drosophila cell lines were obtained, proving to be a powerful tool for the transformation of primary cultures. The expression of the Ras V12 gene promoted cell proliferation by increasing the mitogen-activated protein kinases (MAPK) and by insulin signaling. The control group took 16 to 29 weeks to make the cell confluent, while the introduction of this gene made the cells confluent in three weeks, and allowed for more than 90 duplications, suggesting that the cells became immortal, thus continuous strains (SIMCOX et al., 2008SIMCOX, A. et al. Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes. PLoS Genet, v.4, n.8, 2008. Available from: <journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1000142>. Accessed: Sep. 05, 2020. Epub: 01-August-2008. doi: 10.1371/journal.pgen.1000142.
https://doi.org/10.1371/journal.pgen.100...
).

Cell lines immortalized by Ras V12 activation have increased expression of genes that promote progression in cell cycle and division. The use of this tool in primary cultures of Drosophila embryos, made it possible to obtain cell lines that actively expressed Ras V12 (DEQUÉANT et al., 2015DEQUÉANT, M. L. et al. Discovery of progenitor cell signatures by time-series synexpression analysis during Drosophila embryonic cell immortalization. Proceedings of the National Academy of Sciences, v.112, n.42, p.12974-12979, 2015. Available from: < Available from: https://www.pnas.org/content/112/42/12974 >. Accessed: Nov. 29, 2020. doi: 10.1073/pnas.1517729112.
https://www.pnas.org/content/112/42/1297...
). With its proven efficiency in embryonic Drosophila cells, Ras V12 can be an important tool in immortalizing our primary cultures of Apis mellifera.

Telomerase

Telomerases are ribonucleoproteins that have an RNA component (hTERC) and a catalytic component (hTERT) (HARRINGTON et al., 1997HARRINGTON, L. et al. Human telomerase contains evolutionarily conserved catalytic and structural subunits. Genes & development, v.11, n.23, p.3109-3115, 1997. Available from: <genesdev.cshlp.org/content/11/23/3109>. Accessed: Sep. 12, 2020. doi: 10.1101/gad.11.23.3109.
https://doi.org/genesdev.cshlp.org/conte...
), whose function is to restore base pairs of DNAs lost from telomeres during cell division (MASON et al., 2011MASON, M. et al. Telomerase structure function. Current opinion in structural biology, v.21, n.1, p.92-100, 2011. Available from: <europepmc.org/article/med/21168327>. Accessed: Jul. 21, 2020. doi: 10.1016/j.sbi.2010.11.005.
https://doi.org/10.1016/j.sbi.2010.11.00...
). The length of the chromosomes is restored after each cell division in cells that have active telomerases (ZVEREVA et al., 2010ZVEREVA, M. I. et al.. Telomerase: structure, functions, and activity regulation. Biochemistry (Moscow), v.75, n.13, p.1563-1583, 2010. Available from: <link.springer.com/article/10.1134/S0006297910130055>. Accessed: Jul. 20, 2020. doi: 10.1134/s0006297910130055.
https://doi.org/10.1134/s000629791013005...
). However, this feature is limited to embryonic cells and pluripotent cells. In somatic cells hTERC continues to be expressed, but the expression of the hTERT catalytic component is substantially reduced, losing its function (FORSYTH et al., 2002FORSYTH, N. R. et al. Telomerase and differentiation in multicellular organisms: turn it off, turn it on and turn it off again. Differentiation, v.69, p.188-197, 2002. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/11841477 >. Accessed: Sep. 05, 2019. doi: 10.1046/j.1432-0436.2002.690412.x.
https://www.ncbi.nlm.nih.gov/pubmed/1184...
). With the progressive shortening of the telomeres, the cell reaches its limit of replication and enters senescence (SHAY & WRIGHT, 2000SHAY, J. W.; WRIGHT, W. E. Hayflick, his limit, and cellular ageing. Nature reviews Molecular cell biology, v.1, n.1, p.72-76, 2000. Available from: < Available from: https://www.nature.com/articles/35036093 >. Accessed: Jun. 15, 2020. doi: 10.1038/35036093.
https://www.nature.com/articles/35036093...
). The introduction of hTERT to the genome of a cell appears to result in direct immortalization, since the cell loses its senescence characteristic, thus obtaining the ability to replicate itself indefinitely (SHAY & WRIGHT, 2005SHAY, J. W.; WRIGHT, W. E. Senescence and immortalization: role of telomeres and telomerase. Carcinogenesis, v.26, n.5, p.867-874, 2005. Available from: <Available from: https://academic.oup.com/carcin/article/26/5/867/2390816 >. Accessed: Sep. 09, 2019. doi: 10.1093/carcin/bgh296.
https://academic.oup.com/carcin/article/...
).

The hTERT-BTY cell line was developed from bovine thyroid tissue, using the hTERT gene (MAO et al., 2018MAO, R. et al. Establishment and Evaluation of a Stable Bovine Thyroid Cell Line for Investigating Foot-and-Mouth Disease Virus. Frontiers in Microbiology, v.9, p.2149, 2018. Available from: < Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2018.02149/full >. Accessed: Jul. 20, 2020. doi: 10.3389/fmicb.2018.02149.
https://www.frontiersin.org/articles/10....
). These researchers reported more than 100 passages with the cells maintaining their genotypic and phenotypic characteristics. The insertion of the human hTERT gene allowed the development of the HTERT-AEC II cell line from the bovine alveolar epithelium (SU et al., 2013SU, F. et al. Establishment and evaluation of a stable cattle type II alveolar epithelial cell line. PloS one, v.8, n.9, 2013. Available from: <journals.plos.org/plosone/article?id=10.1371/journal.pone.0076036>. Accessed: Jul. 24, 2020. doi: 10.1371/journal.pone.0076036.
https://doi.org/10.1371/journal.pone.007...
). In addition, there are reports of preliminary studies on the activity of telomerases from a cell line of cockroach tissues (ZHANG et al., 2018ZHANG, X. et al. Establishment of an embryonic cell line from the American cockroach Periplaneta americana (Blattaria: Blattidae) and a preliminary study of telomerase activity changes during the culturing process. In Vitro Cellular & Developmental Biology-Animal, v.54, n.2, p.129-135, 2018. Available from: <link.springer.com/article/10.1007/s11626-017-0223-5>. Accessed: Sep. 19, 2020. doi: 10.1007/s11626-017-0223-5.
https://doi.org/10.1007/s11626-017-0223-...
). These researchers concluded that cells that stabilized in vitro culture had greater telomeric activity, being cultured for three years. In addition to the insertion of genes, the expression of the hTERT gene can be stimulated by chemical carcinogens, through the exposure of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (CHENG et al., 2015CHENG, Y. B. et al. Demethylation of the hTERT promoter in normal human gastric mucosal epithelial cells following N-methyl-N’-nitro-N-nitrosoguanidine exposure. Biomedical Reports, v.3, n.2, p.176-178, 2015. Available from: < Available from: https://www.spandidos-publications.com/br/3/2/176 >. Accessed: Mar. 13, 2020. doi: 10.3892/br.2014.398.
https://www.spandidos-publications.com/b...
).

These studies demonstrate the great importance of telomerases in some insect species, substantiating the possibility of using this agent for cellular immortalization of Apis mellifera.

N-methyl-N-nitro-N-nitrosoguanidine (MNNG)

MNNG is a monofunctional alkylating agent that causes damage through methylation to base pairs of DNAs, has mutagenic capacity and has been used since 1960 (GICHNER & VELEMÍNSKÝ, 1982GICHNER, T.; VELEMÍNSKÝ, J. Genetic effects of N-methyl-N′-nitro-N-nitrosoguanidine and its homologs. Mutation Research/Reviews in Genetic Toxicology, v.99, n.2, p.129-242, 1982. Available from: < Available from: https://www.sciencedirect.com/science/article/abs/pii/0165111082900574?via%3Dihub >. Accessed: Jul. 15, 2020. doi: 10.1016/0165-1110(82)90057-4.
https://www.sciencedirect.com/science/ar...
). Its action on cell transformation is related to several genetic and epigenetic changes (ZHANG et al., 2009ZHANG, B. et al. Altered gene expression and miRNA expression associated with cancerous IEC-6 cell transformed by MNNG. Journal of Experimental & Clinical Cancer Research, v.28, n.1, p.56, 2009. Available from: <link.springer.com/article/10.1186/1756-9966-28-56>. Accessed: Jul. 21, 2020. doi: 10.1186/1756-9966-28-56.
https://doi.org/10.1186/1756-9966-28-56....
). Its carcinogenic effect has been used as a cell-transforming agent in different species for some decades, both in vivo and in vitro (OGURA & FUJIWARA, 1987OGURA, H.; FUJIWARA, T. Establishment and characterization of a virus-free chick cell line. Acta Medica Okayama, v.41, n.3, p.141-143, 1987. Available from: <europepmc.org/article/med/3115063>. Accessed: Jun. 22, 2020. doi: 10.18926/amo/31758.
https://doi.org/10.18926/amo/31758....
; NARESSE et al., 2009NARESSE, L. E. et al. Carcinogenesis of the upper gastrointestinal tract induced by N-methyl-N’-nitro-nitrosoguanidine and reflux of duodenal contents in the rat. Acta cirurgica brasileira, v.24, n.2, p.112-117, 2009. Available from: <scielo.br/scielo.php?pid=S0102-86502009000200007&script=sci_arttext>. Accessed: Sep. 11, 2020. doi: 10.1590/S0102-86502009000200007.
https://doi.org/10.1590/S0102-8650200900...
). The treatment with MNNG in vitro in different human strains and in vivo in rats, demonstrated direct activation of Ras oncogenes, a genetic alteration of great importance in carcinogenesis and cell transformation (KANEKO et al., 2002KANEKO, M. et al. Different genetic alterations in rat forestomach tumors induced by genotoxic and non-genotoxic carcinogens. Carcinogenesis, v.23, n.10, p.1729-1735, 2002. Available from: <academic.oup.com/carcin/article/23/10/1729/2896660>. Accessed: Mar. 14, 2020. doi: 10.1093/carcin/23.10.1729.
https://doi.org/10.1093/carcin/23.10.172...
; LEE et al., 2007LEE, S. H. et al. Chemical Carcinogen, N-methyl-N’-nitro-N-nitrosoguanidine, is Specific Activator of Oncogenic Ras. Cell Cycle, v.6, n.10, p.1257-1264, 2007. Available from: <tandfonline.com/doi/abs/10.4161/cc.6.10.4243>. Accessed: Sep. 28, 2020. doi: 10.4161/cc.6.10.4243.
https://doi.org/10.4161/cc.6.10.4243....
). Treatment in human intestinal mucosal epithelial cells caused demethylation of hTERT reverse transcriptase precursor genes. These mutations can impact malignancy or cell transformation (CHENG et al., 2015CHENG, Y. B. et al. Demethylation of the hTERT promoter in normal human gastric mucosal epithelial cells following N-methyl-N’-nitro-N-nitrosoguanidine exposure. Biomedical Reports, v.3, n.2, p.176-178, 2015. Available from: < Available from: https://www.spandidos-publications.com/br/3/2/176 >. Accessed: Mar. 13, 2020. doi: 10.3892/br.2014.398.
https://www.spandidos-publications.com/b...
).

The use of this agent is not limited to vertebrate cells. Currently, cell lines of insects immortalized with MNNG are already reported. From primary ovarian cultures of pupae of the species Ostrinia furnacalis, two cell lines were developed, called IOZCAS-Osfu-1 and IOZCAS-Osfu-2. After treatment with MNNG, both strains were cultured for 30 passages, successfully cryopreserved and thawed (ZHANG et al., 2014ZHANG, N. et al. A new insect cell line from the pupal ovary of the Asian corn borer moth Ostrinia furnacalis. In Vitro Cellular & Developmental Biology-Animal, v.50, n.3, p.171-173, 2014. Available from: <pubmed.ncbi.nlm.nih.gov/24163160/>. Accessed: Jun. 19, 2020. doi: 10.1007/s11626-013-9704-3.
https://doi.org/10.1007/s11626-013-9704-...
). Another cell line was developed from primary ovary cultures of pupae of the species Spodoptera exígua, which, after treatment with MNNG, showed an increase in cell proliferation and longevity. After characterization, this cell line was called IOZCAS-Spex 12 (LI et al., 2012LI, X. et al. A new insect cell line from pupal ovary of Spodoptera exigua established by stimulation with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). In Vitro Cellular & Developmental Biology-Animal, v.48, n.5, p.271-275, 2012. Available from: <link.springer.com/article/10.1007/s11626-012-9511-2>. Accessed: Jul. 22, 2020. doi: 10.1007/s11626-012-9511-2.
https://doi.org/10.1007/s11626-012-9511-...
). Thus, MNNG demonstrates its capacity for cell transformation, both in mammalian cells and in insect cells. The use for immortalization of primary cultures of Apis mellifera may be just a matter of standardization of the reagents.

CRISPR / Cas9

The Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR / Cas9 was initially discovered as a mechanism of the adaptive immune system of bacteria and Archaes (JANSEN et al., 2002JANSEN, R. et al. Identification of genes that are associated with DNA repeats in prokaryotes. Molecular microbiology, v.43, n.6, p.1565-1575, 2002. Available from: <onlinelibrary.wiley.com/doi/full/10.1046/j.1365-2958.2002.02839.x>. Accessed: Nov. 25, 2020. doi: 10.1046/j.1365-2958.2002.02839.x.
https://doi.org/10.1046/j.1365-2958.2002...
). It is a tool for gene modification that allows targeted deletions, insertions and precise changes in the genome of a large number of organisms and cell types (SANDER & JOUNG, 2014SANDER, J. D.; JOUNG, J. K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature biotechnology, v.32, n.4, p.347-355, 2014. Available from: < Available from: https://www.nature.com/articles/nbt.2842 >. Accessed: Oct. 02, 2020. doi: 10.1038/nbt.2842.
https://www.nature.com/articles/nbt.2842...
). Its use has stood out due to its high specificity, easy handling in vitro and the possibility of simultaneous editing of multiple targets (LINO et al., 2018LINO, C. A. et al. Delivering CRISPR: a review of the challenges and approaches. Drug delivery, v.25, n.1, p.1234-1257, 2018. Available from: <tandfonline.com/doi/full/10.1080/10717544.2018.1474964>. Accessed: Sep. 17, 2020. Epub 25-May-2018. doi: 10.1080/10717544.2018.1474964.
https://doi.org/10.1080/10717544.2018.14...
).

The deletion of the p53 gene in a canine cell culture made it possible to obtain several populations with infinite useful life, resistance to genotoxicity and absence of carcinogenic characteristics (EUN et al., 2019EUN, K. et al. Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research. BMC biotechnology, v.19, n.1, p.1, 2019. Available from: <link.springer.com/article/10.1186/s12896-018-0491-5>. Accessed: Oct. 05, 2020. doi: 10.1186/s12896-018-0491-5.
https://doi.org/10.1186/s12896-018-0491-...
). The inactivation of two loci of CDKN2A, responsible for the expression of p16INK4A and p14ARF, precursors of the activation of tumor suppressors p53 and pRB, associated with ectopic expression of hTERT, was able to immortalize human prostate epithelial cells without any phenotypic changes (ZHAO et al., 2020ZHAO, Z. et al. Immortalization of human primary prostate epithelial cells via CRISPR inactivation of the CDKN2A locus and expression of telomerase. Prostate cancer and prostatic diseases, p.1-11, 2020. Available from: < Available from: https://www.nature.com/articles/s41391-020-00274-4 >. Accessed: Sep. 21, 2020. doi: 10.1038/s41391-020-00274-4.
https://www.nature.com/articles/s41391-0...
). By not directly inactivating the tumor suppressors p53 and pRB, it is expected that other properties of the cells remain normal, such as the characteristic cell phenotype, protein production, and genomic stability (MCKINLEY & CHEESEMAN, 2017MCKINLEY, K. L.; CHEESEMAN, I. M. Large-scale analysis of CRISPR/Cas9 cell-cycle knockouts reveals the diversity of p53-dependent responses to cell-cycle defects. Developmental cell, v.40, n.4, p.405-420, 2017. Available from: <sciencedirect.com/science/article/pii/S1534580717300394>. Accessed: Sep. 29, 2020. doi: 10.1016/j.devcel.2017.01.012.
https://doi.org/10.1016/j.devcel.2017.01...
; ZHAO et al., 2020).

The genome of Africanized bees Apis mellifera has been fully sequenced, enabling biomolecular studies of this species (ELSIK et al., 2014ELSIK, C. G. et al. Finding the missing honey bee genes: lessons learned from a genome upgrade. BMC genomics, v.15, n.1, p.86, 2014. Available from: <link.springer.com/article/10.1186/1471-2164-15-86>. Accessed: Sep. 15, 2020. doi: 10.1186/1471-2164-15-86.
https://doi.org/10.1186/1471-2164-15-86....
; KADRI et al., 2016KADRI, S. M. et al. A variant reference data set for the Africanized honeybee, Apis mellifera. Scientific data, v.3, n.1, p.1-6, 2016. Available from: <nature.com/articles/sdata201697>. Accessed: Oct. 09, 2020. doi: 10.1038/sdata.2016.97.
https://doi.org/10.1038/sdata.2016.97....
). Then, the efficiency of the CRISPR / Cas9 technique was proven in this species of bee, since Mrjp1 (gene of the main protein of royal jelly) was edited and inserted in eggs that generated six genetically modified queens. These queens subsequently oviposited, giving rise to 20 drones, also genetically modified (KOHNO et al., 2016KOHNO, H. et al. Production of Knockout Mutants by CRISPR/Cas9 in the European Honeybee, Apis mellifera L. Zoological science, v.33, n.5, p.505-512, 2016. Available from: <bioone.org/journals/zoological-science/volume-33/issue-5/zs160043/Production-of-Knockout-Mutants-by-CRISPR-Cas9-in-the-European/10.2108/zs160043.full>. Accessed: Oct. 04, 2020. doi: 10.2108/zs160043.
https://doi.org/10.2108/zs160043....
). The editing of genes such as Pax6, a transcriptional regulatory gene in the development of the nervous and ocular system, and Mrjp1 in Apis mellifera with the use of CRISPR / Cas9 demonstrated high efficiency, with editing rates of up to 100% of the samples used (HU et al., 2019HU, X. F. et al. High-efficiency CRISPR/Cas9-mediated gene editing in honeybee (Apis mellifera) embryos. G3: Genes, Genomes, Genetics, v.9, n.5, p.1759-1766, 2019. Available from: <g3journal.org/content/9/5/1759>. Accessed: Oct. 04, 2020. doi: 10.1534/g3.119.400130.
https://doi.org/10.1534/g3.119.400130....
). The efficiency of this technique in editing Apis mellifera genes opens the way for the modification of genes of interest, for cellular immortalization.

With the use of CRISPR / Cas9 to immortalize mammalian cell cultures and demonstrated the efficiency in gene editing in the species of interest, this technique can be of great value in the process of immortalizing bee cells, through the deletion of precursor genes senescence.

CONCLUSION:

Many cell immortalization techniques are currently described for mammals, but they need to be adapted for use in bee cells. The choice of technique and immortalizing agent is fundamental and challenging, because besides the ability to remain viable for countless passages, cells must maintain the genotype and phenotype similar or identical to the original tissue. In this review, some agents for the immortalization of primary cultures were presented, which may be useful in the development of a continuous cell line from Apis mellifera bee cells. However, some techniques or targets of gene editing can generate undesirable changes, which highlights the need for an individual assessment. This assessment should consider the specific agent and the purpose for which the cell line will be used.

ACKNOWLEDGEMENTS

MI Frühauf was the recipient of a Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) fellowship.

REFERENCES

  • BARNES, P. J. et al. Cellular senescence as a mechanism and target in chronic lung diseases. American journal of respiratory and critical care medicine, v.200, n.5, p.556-564, 2019. Available from: < Available from: https://www.atsjournals.org/doi/full/10.1164/rccm.201810-1975TR >. Accessed: May, 17, 2020. doi: 10.1164/rccm.201810-1975TR.
    » https://doi.org/10.1164/rccm.201810-1975TR.» https://www.atsjournals.org/doi/full/10.1164/rccm.201810-1975TR
  • BEAUREPAIRE, A. et al. Diversity and global distribution of viruses of the western honey bee, Apis melífera. Insects, v.11, n.4, p.1-25, 2020. Available from: <Available from: https://www.mdpi.com/2075-4450/11/4/239 >. Accessed: Sep. 14, 2020. doi: 10.3390/insects11040239.
    » https://doi.org/10.3390/insects11040239.» https://www.mdpi.com/2075-4450/11/4/239
  • BRUMBY, A. M.; RICHARDSON, H. E. Scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila. The EMBO journal, v.22, n.21, p.5769-5779, 2003. Available from: < Available from: https://www.embopress.org/doi/full/10.1093/emboj/cdg548 >. Accessed: Jun. 22, 2020. doi: 10.1093/emboj/cdg548.
    » https://doi.org/10.1093/emboj/cdg548.» https://www.embopress.org/doi/full/10.1093/emboj/cdg548
  • CARRILLO-TRIPP, J. et al. In vivo and in vitro infection dynamics of honeybee viruses. Scientific reports, v.6, p.22265, 2016. Available from: <Available from: https://www.nature.com/articles/srep22265 >. Accessed: Nov. 01, 2020. doi: 10.1038/srep22265.
    » https://doi.org/10.1038/srep22265.» https://www.nature.com/articles/srep22265
  • CASTILHO, L., et al. Animal cell technology: from biopharmaceuticals to gene therapy. New York : Garland Science, 2008.
  • CHENG, Y. B. et al. Demethylation of the hTERT promoter in normal human gastric mucosal epithelial cells following N-methyl-N’-nitro-N-nitrosoguanidine exposure. Biomedical Reports, v.3, n.2, p.176-178, 2015. Available from: < Available from: https://www.spandidos-publications.com/br/3/2/176 >. Accessed: Mar. 13, 2020. doi: 10.3892/br.2014.398.
    » https://doi.org/10.3892/br.2014.398.» https://www.spandidos-publications.com/br/3/2/176
  • DANG, C. V. et al. The c-Myc target gene network. Seminars in Cancer Biology, v.16, n.4, p.253-264, 2006. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16904903 >. Accessed: Nov. 05, 2019. doi: 10.1016/j.semcancer.2006.07.014.
    » https://doi.org/10.1016/j.semcancer.2006.07.014.» https://www.ncbi.nlm.nih.gov/pubmed/16904903
  • DEQUÉANT, M. L. et al. Discovery of progenitor cell signatures by time-series synexpression analysis during Drosophila embryonic cell immortalization. Proceedings of the National Academy of Sciences, v.112, n.42, p.12974-12979, 2015. Available from: < Available from: https://www.pnas.org/content/112/42/12974 >. Accessed: Nov. 29, 2020. doi: 10.1073/pnas.1517729112.
    » https://doi.org/10.1073/pnas.1517729112.» https://www.pnas.org/content/112/42/12974
  • EILERS, M.; EISENMAN, R. N. Myc’s broad reach. Genes & Development, v.22, p.2755-2766, 2008. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/18923074 >. Acessed: Nov. 08, 2019. doi: 10.1101/gad.1712408.
    » https://doi.org/10.1101/gad.1712408.» https://www.ncbi.nlm.nih.gov/pubmed/18923074
  • ELSIK, C. G. et al. Finding the missing honey bee genes: lessons learned from a genome upgrade. BMC genomics, v.15, n.1, p.86, 2014. Available from: <link.springer.com/article/10.1186/1471-2164-15-86>. Accessed: Sep. 15, 2020. doi: 10.1186/1471-2164-15-86.
    » https://doi.org/10.1186/1471-2164-15-86.
  • EUN, K. et al. Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research. BMC biotechnology, v.19, n.1, p.1, 2019. Available from: <link.springer.com/article/10.1186/s12896-018-0491-5>. Accessed: Oct. 05, 2020. doi: 10.1186/s12896-018-0491-5.
    » https://doi.org/10.1186/s12896-018-0491-5.
  • FORSYTH, N. R. et al. Telomerase and differentiation in multicellular organisms: turn it off, turn it on and turn it off again. Differentiation, v.69, p.188-197, 2002. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/11841477 >. Accessed: Sep. 05, 2019. doi: 10.1046/j.1432-0436.2002.690412.x.
    » https://doi.org/10.1046/j.1432-0436.2002.690412.x.» https://www.ncbi.nlm.nih.gov/pubmed/11841477
  • GARCÍA-GUTIÉRREZ, L. et al. MYC oncogene contributions to release of cell cycle brakes. Genes, v.10, n.3, p.244, 2019. Available from: < Available from: https://www.mdpi.com/2073-4425/10/3/244 >. Accessed: Jul. 23, 2020. doi: 10.3390/genes10030244.
    » https://doi.org/10.3390/genes10030244.» https://www.mdpi.com/2073-4425/10/3/244
  • GENERSCH, E. et al. Standard methods for cell cultures in Apis mellifera research. Journal of Apicultural Research, v.52, n.1, p.1-8, 2013. Available from: < Available from: https://www.tandfonline.com/doi/abs/10.3896/IBRA.1.52.1.02 >. Accessed: Jun. 24, 2020. doi: 10.3896/IBRA.1.52.1.02.
    » https://doi.org/10.3896/IBRA.1.52.1.02.» https://www.tandfonline.com/doi/abs/10.3896/IBRA.1.52.1.02
  • GICHNER, T.; VELEMÍNSKÝ, J. Genetic effects of N-methyl-N′-nitro-N-nitrosoguanidine and its homologs. Mutation Research/Reviews in Genetic Toxicology, v.99, n.2, p.129-242, 1982. Available from: < Available from: https://www.sciencedirect.com/science/article/abs/pii/0165111082900574?via%3Dihub >. Accessed: Jul. 15, 2020. doi: 10.1016/0165-1110(82)90057-4.
    » https://doi.org/10.1016/0165-1110(82)90057-4.» https://www.sciencedirect.com/science/article/abs/pii/0165111082900574?via%3Dihub
  • GISDER, S.; GENERSCH, E. Viruses of commercialized insect pollinators. Journal of Invertebrate Pathology, v.147, p.51-59, 2017. Available from: <Available from: https://www.sciencedirect.com/science/article/abs/pii/S0022201116300957 >. Accessed: Jul. 12, 2019. doi: 10.1016/j.jip.2016.07.010.
    » https://doi.org/10.1016/j.jip.2016.07.010.» https://www.sciencedirect.com/science/article/abs/pii/S0022201116300957
  • GOBLIRSCH, M. A cell line resource derived from honey bee (Apis mellifera) embryonic tissues. PLoS One, v.8, n.7, 2013. Available from: <journals.plos.org/plosone/article?id=10.1371/journal.pone.0069831>. Accessed: Nov. 15, 2019. doi: 10.1371/journal.pone.0069831.
    » https://doi.org/10.1371/journal.pone.0069831.
  • GRACE, T. D. Establishment of four strains of cells from insect tissues grown in vitro. Nature, v.195, p.788-789, 1962. Available from: <cabdirect.org/cabdirect/abstract/19632900172>. Accessed: Oct. 22, 2019. doi: 10.1038/195788a0.
    » https://doi.org/10.1038/195788a0.
  • GUO, Y. et al. Cell lines for honey bee virus research. Viruses, v.12, n.2, p.236, 2020. Available from: < Available from: https://www.mdpi.com/1999-4915/12/2/236 >. Accessed: Oct. 03, 2020. doi: 10.3390/v12020236.
    » https://doi.org/10.3390/v12020236.» https://www.mdpi.com/1999-4915/12/2/236
  • HARRINGTON, L. et al. Human telomerase contains evolutionarily conserved catalytic and structural subunits. Genes & development, v.11, n.23, p.3109-3115, 1997. Available from: <genesdev.cshlp.org/content/11/23/3109>. Accessed: Sep. 12, 2020. doi: 10.1101/gad.11.23.3109.
    » https://doi.org/genesdev.cshlp.org/content/11/23/3109» https://doi.org/10.1101/gad.11.23.3109.
  • HOLDERFIELD, M. Efforts to develop KRAS inhibitors. Cold Spring Harbor perspectives in medicine, v.8, n.7, 2018. Available from: <perspectivesinmedicine.cshlp.org/content/8/7/a031864>. Accessed: Sep. 14, 2020. doi: 10.1101/cshperspect.a031864.
    » https://doi.org/perspectivesinmedicine.cshlp.org/content/8/7/a031864» https://doi.org/10.1101/cshperspect.a031864.
  • HU, X. F. et al. High-efficiency CRISPR/Cas9-mediated gene editing in honeybee (Apis mellifera) embryos. G3: Genes, Genomes, Genetics, v.9, n.5, p.1759-1766, 2019. Available from: <g3journal.org/content/9/5/1759>. Accessed: Oct. 04, 2020. doi: 10.1534/g3.119.400130.
    » https://doi.org/10.1534/g3.119.400130.
  • JANSEN, R. et al. Identification of genes that are associated with DNA repeats in prokaryotes. Molecular microbiology, v.43, n.6, p.1565-1575, 2002. Available from: <onlinelibrary.wiley.com/doi/full/10.1046/j.1365-2958.2002.02839.x>. Accessed: Nov. 25, 2020. doi: 10.1046/j.1365-2958.2002.02839.x.
    » https://doi.org/10.1046/j.1365-2958.2002.02839.x.
  • JU, H.; GHIL, S. Primary cell culture method for the honeybee Apis mellifera In Vitro Cellular & Developmental Biology-Animal, v.51, n.9, p.890-893, 2015. Available from: <link.springer.com/article/10.1007/s11626-015-9924-9>. Accessed: Oct. 28, 2020. doi: 10.1007/s11626-015-9924-9.
    » https://doi.org/10.1007/s11626-015-9924-9.
  • KABILOVA, T. O. et al. Inhibition of human carcinoma and neuroblastoma cell proliferation by anti-c-myc siRNA. Oligonucleotides, v.16, n.1, p.15-25, 2006. Available from: < Available from: https://www.liebertpub.com/doi/abs/10.1089/oli.2006.16.15 >. Accessed: Sep. 22, 2020. doi: 10.1089/oli.2006.16.15.
    » https://doi.org/10.1089/oli.2006.16.15.» https://www.liebertpub.com/doi/abs/10.1089/oli.2006.16.15
  • KADRI, S. M. et al. A variant reference data set for the Africanized honeybee, Apis mellifera Scientific data, v.3, n.1, p.1-6, 2016. Available from: <nature.com/articles/sdata201697>. Accessed: Oct. 09, 2020. doi: 10.1038/sdata.2016.97.
    » https://doi.org/10.1038/sdata.2016.97.
  • KANEKO, M. et al. Different genetic alterations in rat forestomach tumors induced by genotoxic and non-genotoxic carcinogens. Carcinogenesis, v.23, n.10, p.1729-1735, 2002. Available from: <academic.oup.com/carcin/article/23/10/1729/2896660>. Accessed: Mar. 14, 2020. doi: 10.1093/carcin/23.10.1729.
    » https://doi.org/10.1093/carcin/23.10.1729.
  • KARIM, F. D.; RUBIN, G. M. Ectopic expression of activated Ras1 induces hyperplastic growth and increased cell death in Drosophila imaginal tissues. Development, v.125, n.1, p.1-9, 1998. Available from: <dev.biologists.org/content/125/1/1>. Accessed: May, 18, 2020.
  • KITAGISHI, Y. et al. Long-term cultivation of in vitro Apis mellifera cells by gene transfer of human c-myc proto-oncogene. In Vitro Cellular & Developmental Biology-Animal, v.47, n.7, p.451-453, 2011. Available from: <pubmed.ncbi.nlm.nih.gov/21688083/>. Accessed: Sep. 21, 2019. doi: 10.1007/s11626-011-9431-6.
    » https://doi.org/10.1007/s11626-011-9431-6.
  • KOHNO, H. et al. Production of Knockout Mutants by CRISPR/Cas9 in the European Honeybee, Apis mellifera L. Zoological science, v.33, n.5, p.505-512, 2016. Available from: <bioone.org/journals/zoological-science/volume-33/issue-5/zs160043/Production-of-Knockout-Mutants-by-CRISPR-Cas9-in-the-European/10.2108/zs160043.full>. Accessed: Oct. 04, 2020. doi: 10.2108/zs160043.
    » https://doi.org/10.2108/zs160043.
  • LEE, S. H. et al. Chemical Carcinogen, N-methyl-N’-nitro-N-nitrosoguanidine, is Specific Activator of Oncogenic Ras. Cell Cycle, v.6, n.10, p.1257-1264, 2007. Available from: <tandfonline.com/doi/abs/10.4161/cc.6.10.4243>. Accessed: Sep. 28, 2020. doi: 10.4161/cc.6.10.4243.
    » https://doi.org/10.4161/cc.6.10.4243.
  • LI, X. et al. A new insect cell line from pupal ovary of Spodoptera exigua established by stimulation with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). In Vitro Cellular & Developmental Biology-Animal, v.48, n.5, p.271-275, 2012. Available from: <link.springer.com/article/10.1007/s11626-012-9511-2>. Accessed: Jul. 22, 2020. doi: 10.1007/s11626-012-9511-2.
    » https://doi.org/10.1007/s11626-012-9511-2.
  • LINO, C. A. et al. Delivering CRISPR: a review of the challenges and approaches. Drug delivery, v.25, n.1, p.1234-1257, 2018. Available from: <tandfonline.com/doi/full/10.1080/10717544.2018.1474964>. Accessed: Sep. 17, 2020. Epub 25-May-2018. doi: 10.1080/10717544.2018.1474964.
    » https://doi.org/10.1080/10717544.2018.1474964
  • MAO, R. et al. Establishment and Evaluation of a Stable Bovine Thyroid Cell Line for Investigating Foot-and-Mouth Disease Virus. Frontiers in Microbiology, v.9, p.2149, 2018. Available from: < Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2018.02149/full >. Accessed: Jul. 20, 2020. doi: 10.3389/fmicb.2018.02149.
    » https://doi.org/10.3389/fmicb.2018.02149.» https://www.frontiersin.org/articles/10.3389/fmicb.2018.02149/full
  • MAQSOOD, M. I. et al. Immortality of cell lines: challenges and advantages of establishment. Cell Biology International, v.37, n.10, p.1038-1045, 2013. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/23723166/ >. Accessed: Nov. 01, 2020. doi: 10.1002/cbin.10137.
    » https://doi.org/10.1002/cbin.10137.» https://pubmed.ncbi.nlm.nih.gov/23723166/
  • MASON, M. et al. Telomerase structure function. Current opinion in structural biology, v.21, n.1, p.92-100, 2011. Available from: <europepmc.org/article/med/21168327>. Accessed: Jul. 21, 2020. doi: 10.1016/j.sbi.2010.11.005.
    » https://doi.org/10.1016/j.sbi.2010.11.005.
  • MCKINLEY, K. L.; CHEESEMAN, I. M. Large-scale analysis of CRISPR/Cas9 cell-cycle knockouts reveals the diversity of p53-dependent responses to cell-cycle defects. Developmental cell, v.40, n.4, p.405-420, 2017. Available from: <sciencedirect.com/science/article/pii/S1534580717300394>. Accessed: Sep. 29, 2020. doi: 10.1016/j.devcel.2017.01.012.
    » https://doi.org/10.1016/j.devcel.2017.01.012.
  • NARESSE, L. E. et al. Carcinogenesis of the upper gastrointestinal tract induced by N-methyl-N’-nitro-nitrosoguanidine and reflux of duodenal contents in the rat. Acta cirurgica brasileira, v.24, n.2, p.112-117, 2009. Available from: <scielo.br/scielo.php?pid=S0102-86502009000200007&script=sci_arttext>. Accessed: Sep. 11, 2020. doi: 10.1590/S0102-86502009000200007.
    » https://doi.org/10.1590/S0102-86502009000200007.
  • OGURA, H.; FUJIWARA, T. Establishment and characterization of a virus-free chick cell line. Acta Medica Okayama, v.41, n.3, p.141-143, 1987. Available from: <europepmc.org/article/med/3115063>. Accessed: Jun. 22, 2020. doi: 10.18926/amo/31758.
    » https://doi.org/10.18926/amo/31758.
  • PINTO, L. C. et al. Mebendazole induces apoptosis via C-MYC inactivation in malignant ascites cell line (AGP01). Toxicology in Vitro, v.60, p.305-312, 2019. Available from: <pubmed.ncbi.nlm.nih.gov/31207347/>. Accessed: Jun, 18, 2020. doi: 10.1016/j.tiv.2019.06.010.
    » https://doi.org/10.1016/j.tiv.2019.06.010.
  • PIRES, C. S. S. et al. Weakness and collapse of bee colonies in Brazil: are there cases of CCD?. Pesquisa Agropecuária Brasileira, v.51, n.5, p.422-442, 2016. Available from: <Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-204X2016000500422 >. Accessed: Nov. 04, 2019. doi: 10.1590/S0100-204X2016000500003.
    » https://doi.org/10.1590/S0100-204X2016000500003.» http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-204X2016000500422
  • POPPINGA, Lena et al. Identification and functional analysis of the S-layer protein SplA of Paenibacillus larvae, the causative agent of American Foulbrood of honey bees. PLoS Pathog, v.8, n.5, p.e1002716, 2012. Available from: <Available from: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1002716 >. Accessed: Nov. 01, 2020. doi: 10.1371/journal.ppat.1002716.
    » https://doi.org/10.1371/journal.ppat.1002716.» https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1002716
  • PRIOR, I.A. et al. A comprehensive survey of Ras mutations in cancer. Cancer research, v.72, n.10, p.2457-2467, 2012. Available from: <cancerres.aacrjournals.org/content/72/10/2457>. Accessed: Jun. 20, 2020. doi: 10.1158/0008-5472.can-11-2612.
    » https://doi.org/10.1158/0008-5472.can-11-2612.
  • PRIOR, I. A. et al. The frequency of Ras mutations in cancer. Cancer Research, v.80, n.14, 2020. Available from: <cancerres.aacrjournals.org/content/80/14/2969>. Accessed: Oct. 17, 2020. doi: 10.1158/0008-5472.CAN-19-3682.
    » https://doi.org/10.1158/0008-5472.CAN-19-3682.
  • SANDER, J. D.; JOUNG, J. K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature biotechnology, v.32, n.4, p.347-355, 2014. Available from: < Available from: https://www.nature.com/articles/nbt.2842 >. Accessed: Oct. 02, 2020. doi: 10.1038/nbt.2842.
    » https://doi.org/10.1038/nbt.2842.» https://www.nature.com/articles/nbt.2842
  • SHAY, J. W.; WRIGHT, W. E. Hayflick, his limit, and cellular ageing. Nature reviews Molecular cell biology, v.1, n.1, p.72-76, 2000. Available from: < Available from: https://www.nature.com/articles/35036093 >. Accessed: Jun. 15, 2020. doi: 10.1038/35036093.
    » https://doi.org/10.1038/35036093.» https://www.nature.com/articles/35036093
  • SHAY, J. W.; WRIGHT, W. E. Senescence and immortalization: role of telomeres and telomerase. Carcinogenesis, v.26, n.5, p.867-874, 2005. Available from: <Available from: https://academic.oup.com/carcin/article/26/5/867/2390816 >. Accessed: Sep. 09, 2019. doi: 10.1093/carcin/bgh296.
    » https://doi.org/10.1093/carcin/bgh296.» https://academic.oup.com/carcin/article/26/5/867/2390816
  • SIMANSHU, D. K. et al. RAS proteins and their regulators in human disease. Cell, v.170, n.1, p.17-33, 2017. Available from: < Available from: https://www.sciencedirect.com/science/article/pii/S0092867417306505 >. Accessed: Jun. 26, 2020. doi: 10.1016/j.cell.2017.06.009.
    » https://doi.org/10.1016/j.cell.2017.06.009.» https://www.sciencedirect.com/science/article/pii/S0092867417306505
  • SIMCOX, A. et al. Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes. PLoS Genet, v.4, n.8, 2008. Available from: <journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1000142>. Accessed: Sep. 05, 2020. Epub: 01-August-2008. doi: 10.1371/journal.pgen.1000142.
    » https://doi.org/10.1371/journal.pgen.1000142.
  • STACEY, G. Primary Cell Cultures and Immortal Cell Lines. Encyclopedia of Life Sciences, p.1-5, 2006. Available from: <Available from: https://onlinelibrary.wiley.com/doi/10.1038/npg.els.0002564 >. Accessed: Nov. 04, 2019. doi: 10.1038/npg.els.0002564.
    » https://doi.org/10.1038/npg.els.0002564.» https://onlinelibrary.wiley.com/doi/10.1038/npg.els.0002564
  • SU, F. et al. Establishment and evaluation of a stable cattle type II alveolar epithelial cell line. PloS one, v.8, n.9, 2013. Available from: <journals.plos.org/plosone/article?id=10.1371/journal.pone.0076036>. Accessed: Jul. 24, 2020. doi: 10.1371/journal.pone.0076036.
    » https://doi.org/10.1371/journal.pone.0076036.
  • VAFA, O. et al. c-Myc can induce DNA damage, increase reactive oxygen species, and mitigate p53 function. A mechanism for oncogene-induced genetic instability. Molecular Cell, v.9, n.5, p.1031-1044, 2002. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/12049739 >. Accessed: Nov. 12, 2019. doi: 10.1016/s1097-2765(02)00520-8.
    » https://doi.org/10.1016/s1097-2765(02)00520-8.» https://www.ncbi.nlm.nih.gov/pubmed/12049739
  • VANENGELSDORP, D. et al. Colony Collapse Disorder (CCD) and bee age impact honey bee pathophysiology. PLoS One, v.12, n.7, e0179535, 2017. Available from: <journals.plos.org/plosone/article?id=10.1371/journal.pone.0179535>. Accessed: Oct. 11, 2019. doi: 10.1371/journal.pone.0179535.
    » https://doi.org/10.1371/journal.pone.0179535.
  • VIRGIN, H. W. et al. Redefining chronic viral infection. Cell, v.138, n.1, p.30-50, 2009. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S0092867409007831 >. Accessed: Nov. 17, 2020. doi: 10.1016/j.cell.2009.06.036.
    » https://doi.org/10.1016/j.cell.2009.06.036.» https://www.sciencedirect.com/science/article/pii/S0092867409007831
  • WENNERBERG, K. et al. The Ras superfamily at a glance. Journal of cell science, v.118, n.5, p.843-846, 2005. Available from: <Available from: https://jcs.biologists.org/content/118/5/843 >. Accessed: Jul. 11, 2020. doi: 10.1242/jcs.01660.
    » https://doi.org/10.1242/jcs.01660.» https://jcs.biologists.org/content/118/5/843
  • WHITE, M. A. et al. Multiple ras functions can contribute to mammalian cell transformation. Cell, v.80, n.4, p.533-541, 1995. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/0092867495905073 >. Accessed: Nov. 07, 2019. doi: 10.1016/0092-8674(95)90507-3.
    » https://doi.org/10.1016/0092-8674(95)90507-3.» https://www.sciencedirect.com/science/article/pii/0092867495905073
  • WOLPAW, A. J.; DANG, C. V. MYC-induced metabolic stress and tumorigenesis. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer, v.1870, n.1, p.43-50, 2018. Available from: <pubmed.ncbi.nlm.nih.gov/29791870/>. Accessed: Oct. 12, 2020. doi: 10.1016/j.bbcan.2018.05.003.
    » https://doi.org/10.1016/j.bbcan.2018.05.003.
  • WRIGHT, W. E.; SHAY, J. W. The two-stage mechanism controlling cellular senescence and immortalization. Experimental Gerontology, v.27, p.383-389, 1992. Available from: <europepmc.org/abstract/med/1333985>. Accessed: Nov. 06, 2019. doi: 10.1016/0531-5565(92)90069-c.
    » https://doi.org/10.1016/0531-5565(92)90069-c.
  • ZHANG, N. et al. A new insect cell line from the pupal ovary of the Asian corn borer moth Ostrinia furnacalis. In Vitro Cellular & Developmental Biology-Animal, v.50, n.3, p.171-173, 2014. Available from: <pubmed.ncbi.nlm.nih.gov/24163160/>. Accessed: Jun. 19, 2020. doi: 10.1007/s11626-013-9704-3.
    » https://doi.org/10.1007/s11626-013-9704-3.
  • ZHANG, B. et al. Altered gene expression and miRNA expression associated with cancerous IEC-6 cell transformed by MNNG. Journal of Experimental & Clinical Cancer Research, v.28, n.1, p.56, 2009. Available from: <link.springer.com/article/10.1186/1756-9966-28-56>. Accessed: Jul. 21, 2020. doi: 10.1186/1756-9966-28-56.
    » https://doi.org/10.1186/1756-9966-28-56.
  • ZHANG, X. et al. Establishment of an embryonic cell line from the American cockroach Periplaneta americana (Blattaria: Blattidae) and a preliminary study of telomerase activity changes during the culturing process. In Vitro Cellular & Developmental Biology-Animal, v.54, n.2, p.129-135, 2018. Available from: <link.springer.com/article/10.1007/s11626-017-0223-5>. Accessed: Sep. 19, 2020. doi: 10.1007/s11626-017-0223-5.
    » https://doi.org/10.1007/s11626-017-0223-5.
  • ZHAO, Z. et al. Immortalization of human primary prostate epithelial cells via CRISPR inactivation of the CDKN2A locus and expression of telomerase. Prostate cancer and prostatic diseases, p.1-11, 2020. Available from: < Available from: https://www.nature.com/articles/s41391-020-00274-4 >. Accessed: Sep. 21, 2020. doi: 10.1038/s41391-020-00274-4.
    » https://doi.org/10.1038/s41391-020-00274-4.» https://www.nature.com/articles/s41391-020-00274-4
  • ZVEREVA, M. I. et al.. Telomerase: structure, functions, and activity regulation. Biochemistry (Moscow), v.75, n.13, p.1563-1583, 2010. Available from: <link.springer.com/article/10.1134/S0006297910130055>. Accessed: Jul. 20, 2020. doi: 10.1134/s0006297910130055.
    » https://doi.org/10.1134/s0006297910130055.
  • CR-2020-1111.R1

Publication Dates

  • Publication in this collection
    26 July 2021
  • Date of issue
    2021

History

  • Received
    23 Dec 2020
  • Accepted
    16 Mar 2021
  • Reviewed
    04 May 2021
Universidade Federal de Santa Maria Universidade Federal de Santa Maria, Centro de Ciências Rurais , 97105-900 Santa Maria RS Brazil , Tel.: +55 55 3220-8698 , Fax: +55 55 3220-8695 - Santa Maria - RS - Brazil
E-mail: cienciarural@mail.ufsm.br