CC BY
23
FULL COMMUNICATION
CYTOGENETICS
Molecular cytogenetic characterization of two murine cancer cell lines derived from salivary gland
Ralf Steinacker1, Thomas Liehr1, Nadezda Kosyakova1, Martina Rincic2, and Shaymaa S. Hussein Azawi1
1Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Am Klinikum 1, D-07747 Jena, Germany
2Department for Functional Genomics, Centre for Translational and Clinical Research, University Hospital Centre Zagreb, University of Zagreb School of Medicine, Zagreb, Croatia
Address correspondence and requests for materials to Thomas Liehr, [email protected]
Abstract
Here two murine salivary gland cancer (SGC) cell lines WR21 and SCA-9 were studied for the first time in detail by high-resolution molecular cytogenetic approaches. This study revealed that these cell lines are models for human SGCs of initial stage myoepithelioid or mucoepidermoid (WR21) and of advanced stage mucoepidermoid (SCA-9) tumors. Besides, three genes most likely playing a role in SGC development (FGF10, ELAVL1/HUR and SEL1) were identified. All of them were involved in translocation events in these in vitro models and thus were most likely activated. Overall, the present study highlights the necessity not only to establish but also to genetically characterize murine tumor cell lines. Without such a characterization they cannot be used in a reasonable way in research.
Keywords: salivary gland cancer (SGC), murine tumor cell lines, WR21, SCA-9, myoepithelioid SGC, mucoepidermoid SGC, FGF10, ELAVL1/HUR, SEL1.
Introduction
Citation: Steinacker, R., Liehr, T., Kosyakova, N., Rincic, M., and Hussein Azawi, S. S. 2018. Molecular cytogenetic characterization of two murine cancer cell lines derived from salivary gland. Bio. Comm. 63(4): 243-255. https://doi. org/10.21638/spbu03.2018.403
Author's information: Ralf Steinacker; Thomas Liehr, PD, Dr., PhD, Head of Laboratory, orcid.org/0000-0003-1672-3054; Nadezda Kosyakova, MD, PhD, Scientist; Martina Rincic, PhD, Scientist; Shaymaa S. Hussein Azawi, MSc, PhD Student
Manuscript Editor: Alla Krasikova, Saint Petersburg State University, Saint Petersburg, Russia
Received: October 30, 2018;
Revised: November 22, 2018;
Accepted: December 28, 2018;
Copyright: © 2018 Liehr et al. This is an open-access article distributed under the terms of the License Agreement with Saint Petersburg State University, which permits to the authors unrestricted distribution, and self-archiving free of charge.
Funding: This work was supported by Grant No. 2013.032.1 of the Wilhelm Sander-Stiftung.
Competing interests: The authors have declared that no competing interests exist.
Salivary gland cancers (SGCs) are a specific and rare subgroup of tumors known from oral and maxillofacial clinical practice. They account for around 3-5 % of all head and neck cancers and for only less than 0.5 % of all cancers (Zboray et al., 2018). Nevertheless, SGCs include more than 35 histological subtypes and are known for their progressive and heterogeneous clinical behavior (El-Naggar et al., 1997; Persson et al., 2009; Cao et al., 2018).
The mentioned subtypes of SGCs exist partially due to the fact that salivary glands consist of three major paired glands (parotid, submandibular and sublingual) and minor glands, located in the mucosa of the palate, lips and respiratory tract (Omitola and Iyogun, 2018; Zboray et al., 2018). Tumors located in the parotid part are only 25 % malignant. However, the incidence of malignancies is much higher in the submandibular part (50 %) and minor salivary glands (60-80 %) (Sood et al., 2016; Solanki, 2011). In general, SGCs are divided in two forms: (i) a simple palpable lump, being well-defined, discrete, and mobile, and (ii) a lump with significant accompanying symptoms like pain, rapid growth, fixity to surrounding structures, nerve involvement and/or neck metastasis (Sood et al., 2016). Current therapeutic options for human SGCs are limited. Depending on their location, some SGCs can be surgically addressed, while others are difficult to remove completely. Radiation therapy is used; still it has turned out to be less effective for clinical treatment. Accordingly, chemotherapy is the only treatment option in metastatic SGCs (Keller et al., 2017; Zboray et al., 2018).
The detailed molecular mechanisms controlling tumor progression and metastasis in SGCs and their genetic profiles are still not well understood (Vekony et al., 2009). However, characterization of these underlying mechanisms is essential for understanding and development of more effective methods of diagnosis and treatment against the disease (Murase et al., 2016). Hence, there is a need for better understanding of the genetics and molecular mechanisms of SGC-pathogenesis, to be used in the future toward the development of novel therapeutic approaches (Cao et al., 2018; Murase et al., 2016; Zboray et al., 2018).
One possible approach for studying the biology of SGCs and developing new therapeutic strategies is the use of mouse models (Zboray et al., 2018). Previous research showed that submandibular gland-derived tumor cell lines present characteristics of differentiated epithelial cells and can be used to study proliferation signaling pathways and their regulation (Trzaskawka et al., 2000; Español et al., 2012). Still, surprisingly, murine cell lines used as models for human SGCs have not yet been very well characterized genetically for their tumor-associated alterations.
Fluorescence in situ hybridization (FISH) and microarray-based comparative genomic hybridization (array CGH) were used in this study to determine the genomic aberrations of the two murine SGC cell lines WR21 and SCA-9. WR21 was first described by Young et al. (2006) as being derived from a salivary tumor in a male wap-ras subline 69-2 (C57BL/6, SJL) transgenic mouse; it was already established in 1989. Such tumors are described as extremely aggressive and as expressing high levels of oncogenic ras-protein from the activated human H-RAS transgene (Nielsen et al., 1994). SCA-9 was already established in 1980 (Barka, 1980; Barka et al., 2005) and was derived from a carcinogen (7,12-dimethylbenz(a)anthracene) -induced tumor of a male Swiss-Webster mouse submandibular gland. Both cell lines have only been applied in 10 published studies (see Pubmed: https://www.ncbi.nlm.nih.gov/ pubmed/?term=wr21 and https://www.ncbi.nlm.nih. gov/pubmed/?term=SCA-9+mouse), which may also be due to the fact that their genetics had not been studied in detail. Here, we analyzed WR21 and SCA-9 cell lines by FISH-banding and aCGH and aligned them with their human SGC subtypes.
Material and Methods
CELL LINES
The cell lines WR21 and SCA-9 were obtained from American Type Culture Collection (ATCC® CRL-2189™, ATCC® CRL-1734™; Middlesex, Uk). They are indicated there as 'not further characterized salivary tumor lines'
to be grown adherently in DMEM medium containing 10 % fetal calf serum in the presence of antibiotics. For this study, cells were worked up cytogenetically as previously reported (Rhode et al., 2018) and in parallel whole genomic DNA was extracted using the Blood & Cell Culture DNA Midi Kit (Qiagen; Hilden, Germany) according to the manufacturer's instructions and described elsewhere (Kubikova et al., 2017). We conducted molecular cytogenetic analyses on the cell line-derived chromosomes and aCGH analyses on the extracted DNA (see below).
According to the ethical committee (medical faculty) and the Animal Experimentation Commission of the Friedrich Schiller University, there are no ethical agreements necessary for studies involving murine tumor cell lines like WR21 and SCA-9.
MOLECULAR CYTOGENETICS
FISH was performed as previously described (Kubikova et al., 2017). Whole chromosome paints ("SkyPaint™ DNA Kit M-10 for Mouse Chromosomes", Applied Spectral Imaging, Edingen-Neckarhausen, Germany) were used for multicolor-FISH (mFISH), and murine chromosome-specific multicolor banding (mcb) probe mixes for FISH-banding (Liehr et al., 2006). At least 30 metaphases were documented and analyzed for each probe set ((including using SkyPaintTM) Zeiss Axioplan microscopy, equipped with ISIS software (MetaSystems, Altlussheim, Germany)). Array-based comparative ge-nomic hybridization (aCGH) was done according to standard procedures by "SurePrint G3 Mouse CGH Mi-croarray, 4x180K" (Agilent Technologies) (Kubikova et al., 2017).
DATA ANALYSIS
The breakpoints and imbalances of WR21 and SCA-9 were determined after analyses of aCGH and mcb data, and aligned to human homologous regions using En-sembl and the UCSC Genome Browser, as previously described (Leibiger et al., 2013). The obtained data were compared to genetic changes known from human SGCs according to Fowler et al. (2006), Rao et al. (2008), Pers-son et al. (2009), Vekony et al. (2009), Jee et al. (2013) and Matse et al. (2017).
Results
WR21 is mitotically relatively stable, and the near-diploid karyotype has an overall low rate of single cell aberrations; still, it developed two main clones. Clone 1 represents 16.6 % of the cells and has the karyotype 44,XY,+Y,+inv(4)(A1C4),+8,der(9)(A1->E2::E1-qter),der(12)(A1->F2::F1->qter),+19. Clone 2 together with one subclone was found in 83.4 % of the cells. Main-
Fig. 1. Murine multicolor banding (mcb) was applied on chromosomes of SGC cell line WR21. Typical pseudocolor banding for all 21 different murine chromosomes is shown for clone 2. This figure depicts the summary of 21 chromosome-specific FISH-experiments. One derivative chromosome consisting of two different chromosomes is highlighted by frames and shown twice in this summarizing karyogram.
Fig. 2. mcb-results for SGC cell line SCA-9 cell line are shown. Four derivative chromosomes are highlighted by blue frames and shown twice in this summarizing karyogram; the derivative chromosome 5 is highlighted by a yellow frame.
Table 1. Imbalances larger than one cytoband present in WR21 were translated to their corresponding homologous regions in human karyotype (see Suppl. 1) and are listed in the first column. Those are compared to four common types of human SGCs
Chr. region in human WR21 Adenoid cystic carcinoma Myoepitheliomas pleomorphic adenoma Mucoepidermoid
11q13.3 Gain Gain
19p13.2-p13.12 Gain Gain
11q12.1-q13.3 Gain Gain Gain Gain
8p23.3-p21.3 Gain Gain Loss Loss
13q33.1-q34 Gain Gain Gain
22q12.3 Gain Gain
8p12-p11.21 Gain Gain
2q22.1-q32.1 Loss Gain
19p13.12-p13.11 Gain Gain
3q25.1-q26.2 Gain Gain
Overall agreement 3/10 4/10 1/10 4/10
Table 2. Imbalances larger than one cytoband present in SCA-9 were translated to their corresponding homologous regions in human karyotype (see Suppl. 2) and are listed in the first column. Those are compared to 4 human SCG-subtypes
Chr. region in human SCA-9 copy numbers Adenoid cystic carcinoma Myoepitheliomas pleomorphic adenoma Mucoepidermoid
16p altered Gain Gain
9q33.3-q34.3 altered Gain
11q23.3 altered Gain
19q13.3-p13.11 altered Gain Gain
21q22.3 altered Gain
13q21-q22 altered Loss Gain Gain
1p32-p36 altered Loss Loss
5q13.2-q15 altered Loss
3p21.3 altered Loss
11pter-p14.3 altered Loss
15q25~qter altered Gain
6p22~q24 altered Gain
5pter-p15.31 altered Gain
9q33.3-q34.3 altered Gain
18q12.2-qter altered Gain
12p13.2 altered Loss
Overall agreement 7/16 1/16 2/16 11/16
clone of clone 2 revealed the karyotype 43,XY)+Y,inv(4) (A1C4),+der(4)t(4;8)(:4C4->4A4::A1-E2),+19 and was present in 77 % of all cells (Fig. 1). In the remaining 6.4 % of the cells, clone 2 further acquired a loss in one of the Y-chromosomes; i.e., there was a del(Y) — this subset was called clone 2a.
In SCA-9 most chromosomes are tetraploid and the cell line is chromosomally instable, as reflected by many single cell aberrations: 62~76,XX,-1,-2,idic(4)(A1;A1),del(4) (C1),del(4)(C1),der(5)(A1->G3::G3->G2:),der(5)t(5;8) (5A1->5G3::5G3->5G2::8B1->8E2),+6,-7,-8,-8,-9,-12,-12,-13,der(14)t(14;8)(C1;A1)x2, der(18)t(18;12)(D;E),-18
Fig. 3. Imbalances present in WR21 are summarized with respect to a diploid basic karyotype. Gains are depicted as green bars, losses as red bars. In upper part the results are shown for the murine karyotype and in the lower part the translation to human genome. The dark green labeled region of gain at murine chromosome 4 was not detected in aCGH and also not translated to human karyotype.
Most data from the mFISH and mcb for the WR21 and SCA-9 (Table 1) agreed with the aCGH data; the results are shown in Figs. 3 and 4. Some small deletions in murine chromosome 2 and gains in murine chromosomes 3 and 4 (here 4A4 to 4C4, clearly visible mcb) were missed in aCGH. These results were translated to the human genome in the same figures. For this study, imbalances larger than 3.5 megabase pairs were included in the evaluation.
According to the corresponding homologous regions in the human karyotype, we compared the results for both cell lines (Table 1 and 2) with the imbalances for four (adenoid cystic carcinoma, myoepitheliomas, pleomorphic adenoma and mucoepidermoid) of the most common types of SGCs. The highest concordance was found with SGCs of myoepitheliomas and mucoepidermoid for WR21, and of mucoepidermoid for SCA-9.
Fig. 4. Imbalances present in SCA 9 are summarized with respect to a tetraploid basic karyotype. Remainder figure as described in legend for Fig. 3.
Discussion
Both the low incidence and heterogeneity of pathology in SGCs explain why this tumor is one of the least studied human cancer types (Seethala, 2017). SGCs present a diverse range of histological and clinical characteristics (Sood et al., 2018). In the literature, there is also significant heterogeneity in the aberrant genetic and molecular pathways described contributing to the development of SGCs (Müller, 2013; Yin and Ha, 2016).
The two murine tumor cell lines which we examined, commercially available as model systems for SGCs, were successfully studied using molecular cytogenetics (mFISH, mcb and aCGH) to provide a comprehensive cytogenetic description regarding ploidy, numerical and structural aberrations and tumor-associated breakpoints, as previously done for other murine tumor cell lines (Leibiger et al., 2013; Kubicova et al., 2017; Guja et al., 2017; Rhode et al., 2018).
In our results, clonal changes with few aberrations from the main clone were observed in both cell lines,
even though two small subclones (denominated as 1 and 2a) were characterizable in WR21 besides one mainline (clone 2). As these cell lines were not karyotyped at the time of establishing, nothing can be stated about karyo-typic evolution since then. Considering our own previous studies in other, several decades-old cell lines with known original chromosomal content (Leibiger et al., 2013; Kubicova et al., 2017; Guja et al., 2017; Rhode et al., 2018), all of those were surprisingly stable compared to the original description of the chromosome sets.
Overall, WR21 showed clearly a less aberrant karyotype than SCA-9. Compared to most other solid epithelial tumors, SGCs often were correlated with a normal karyotype or small numbers of chromosomal aberrations (Martins et al., 1995; El Naggar et al., 1997; Hungermann et al., 2002; Vekony et al., 2009), and as expected, salvia carcinomas displayed more chromosomal events than benign tumors from this tissue (Ve-kony et al., 2009). Thus, WR21 may represent a benign or less advanced cell line than SCA-9. This view is also supported by the fact that polyploidization, i.e. a basic tetraploid karyotype, was observed only in SCA-9, while WR21 was diploid with a gain of only three (derivative) chromosomes. This could be related to telomere-driven tetraploidization in the context of tumor progression (Davoli and de Lange, 2012), but could also be just a cell culture effect (Mastromonaco et al., 2006).
Gains of copy number were observed in both studied cell lines; while gains were more frequent than loss for WR21, the data interpretation used for SCA-9 seems to show a loss rather than a gain of copy numbers. However, this is due to the fact that SCA-9 was interpreted as basically tetraploid — so the high frequency of losses as given in Fig. 4 must consider that this cell line has a massive gain of copy numbers along the entire genome, and the summary in Fig. 4 rather highlights the genomic instability of this cell line.
According to the characterization of WR21 and SCA-9, neither cell line is a model for adenoid cystic carcinomas or pleomorphic adenomas, but most likely for mucoepidermoid SGCs (SCA-9, WR21) and/or myoepitheliomas (WR21 — see Tabs. 1-2).
Interestingly, the breakpoint 13D2 observed in WR21 comprises the gene FGF10 (see suppl. Tab. 1), which has been associated with salivary gland development (Krejci et al., 2009) and breast cancer (Itoh and Ohta, 2014). Maybe this is a hint that this gene also plays a role in SGCs. For SCA-9 similarly a breakpoint in 8A1 could be associated with the gene ELAVL1, also called HUR, being described as playing a role in salvia metabolism (Palanisa-my et al., 2008) and mucoepidermoid SGC (Cho et al., 2007). The latter confirms that SCA-9 is a model for advanced mucoepidermoid SGC. Another breakpoint (12E) including gene SEL1 plays a role in the salivary glands of Sjogren's syndrome patients (Barrera et al., 2016).
In conclusion, the present study narrowed down the subtypes of two long established murine cancer cell lines to SGCs of mucoepidermoid (SCA-9, WR21) and/ or myoepithelioma (WR21) subtypes and identified three oncogenes potentially playing a role in SGC development. FGF10, ELAVL1/HUR and SEL1 should be further studied with special attention in SGCs. The chromosomal content of the cells should certainly be controlled before doing extensive further studies, to exclude studying subclones with potentially different and/ or advanced karyotypic evolution.
References
Barrera, M.J., Aguilera, S., Castro, I., Cortés, J., Baham-ondes, V., Quest, A. F.G., Molina, C., González, S., Hermoso, M., Urzúa, U., Leyton, C. and González, M.J. 2016. Pro-inflammatory cytokines enhance ERAD and ATF6a pathway activity in salivary glands of Sjogren's syndrome patients. J Autoimmun 75(1):68-81. https://doi. org/10.1016/j.jaut.2016.07.006 Barka, T. 1980. Biologically active polypeptides in submandibular glands,J Histochem Cytochem 28(8):836-859. https:// doi.org/10.1177/28.8.7003006 Barka, T., Gresik, E.S. and Miyazaki, Y. 2005. Differentiation of a mouse submandibular gland-derived cell line (SCA) grown on matrigel. Exp Cell Res 308(2):394-406. https:// doi.org/10.1016/j.yexcr.2005.04.025 Cao, Y., Liu, H., Gao, L., Lu, L., Du, L., Bai, H., Li, J., Said, S., Wang, X., Song, J., Serkova, N., Wei, M., Xiao, J. and Lu, S. 2018. Cooperation between pten and smad4 in murine salivary gland tumor formation and progression. Neoplasia 20:764-774. https://doi.org/10.101 6/j. neo.2018.05.009 Cho, N. P., Han, H.S., Soh, Y. and Son, H.J. 2007. Overexpression of cyclooxygenase-2 correlates with cytoplasmic HuR expression in salivary mucoepidermoid carcinoma but not in pleomorphic adenoma. J Oral Pathol Med 36(5):297-203. https://doi.org/10.1111/j.1600-0714.2007.00526.x Davoli, T. and de Lange, T. 2012. Telomere-driven tetraploidi-zation occurs in human cells undergoing crisis and promotes transformation of mouse cells. Cancer Cell 21(6):765-776. https://doi.org/10.1016/j.ccr.2012.03.044 El-Naggar, A.K., Dinh, M., Tucker, S.L., Gillenwater, A., Luna, M.A. and Batsakis, J.G. 1997. Chromosomal and DNA ploidy characterization of salivary gland neoplasms by combined FISH and flow cytometry. Hum Pathol 28(8):881-886. https://doi.org/10.1016/S0046-8177(97)90001-0 Español, A., Dasso, M., Cella, M., Goren, N. and Sales, M.E. 2012. Muscarinic regulation of SCA-9 cell proliferation via nitric oxide synthases, arginases and cyclooxygen-ases. Role of the nuclear translocation factor-KB. Eur J Pharmacol 683(1-3):43-53. https://doi.org/10.1016/j. ejphar.2012.03.013 Fowler, M.H., Fowler, J., Ducatman, B., Barnes, L. and Hunt, J. L. 2006. Malignant mixed tumors of the salivary gland: a study of loss of heterozygosity in tumor suppressor genes. Mod Pathol 19(3):350-355. https://doi. org/10.1038/modpathol.3800533 Guja, K., Liehr, T., Rincic, M., Kosyakova, N. and Hussein Azawi, S.S. 2017. Molecular cytogenetic characterization identified the murine B-cell lymphoma cell line A-20 as a model for sporadic Burkitt's lymphoma.
J Histochem Cytochem 65(11):669-677. https://doi. org/10.1369/0022155417731319 Hungermann, D., Roeser, K., Buerger, H., Jakel, T., Loening, T. and Herbst, H. 2002. Relative paucity of gross genetic alterations in myoepitheliomas and myoepithelial carcinomas of salivary glands. J Pathol 198(4):487-494. https:// doi.org/10.1002/path.1234 Itoh, N. and Ohta, H. 2014. Fgf10: a paracrine-signaling molecule in development, disease, and regenerative medicine. Curr Mol Med 14(4):504-509. https://doi.org/10.217 4/1566524014666140414204829 Jee, K.J., Persson, M., Heikinheimo, K., Passador-Santos, F., Aro, K., Knuutila, S., Odell, E. W., Mäkitie, A., Sundelin, K., Stenman, G. and Leivo, I. 2013. Genomic profiles and CRTC1-MAML2 fusion distinguish different subtypes of mucoepidermoid carcinoma. Mod Pathol 26(2):213-222. https://doi.org/10.1038/modpathol.2012.154 Keller, G., Steinmann, D., Quaas, A., Grünwald, V., Janssen, S. and Hussein, K. 2017. New concepts of personalized therapy in salivary gland carcinomas. Oral Oncol 68(1):103-113. https://doi.org/10.1016/j.oraloncology.2017.02.018 Krejci, P., Prochazkova, J., Bryja, V., Kozubik, A. and Wilcox, W.R. 2009. Molecular pathology of the fibroblast growth factor family. Hum Mutat 30(9):1245-1255. https://doi.org/10.1002/humu.21067 Kubicova, E., Trifonov, V., Borovecki, F., Liehr, T., Rincic, M., Kosyakova, N. and Hussein, S.S. 2017. First molecular cytogenetic characterization of murine malignant mesothelioma cell line AE17 and in silico translation to the human genome. Curr Bioinform 12(1):11-18. https://doi. org/10.2174/1574893611666160606164459 Leibiger, C., Kosyakova, N., Mkrtchyan, H., Glei, M., Trifonov, V. and Liehr, T. 2013. First molecular cytogenetic high resolution characterization of the NIH 3T3 cell line by murine multicolor banding.J Histochem Cytochem 61(4):306-312. https://doi.org/10.1369/0022155413476868 Liehr, T., Starke, H., Heller, A., Kosyakova, N., Mrasek, K., Gross, M., Karst, C., Glaser, M., Fickelscher, I., Kue-chler, A., Trifonov, V., Romanenko, S.A. and Weise, A. 2006. Multicolor fluorescence in situ hybridization (FISH) applied to FISH-banding. Cytogenet Genome Res 114(3-4):240-244. https://doi.org/10.1 159/000094207 Mastromonaco, G.F., Perrault, S.D., Betts, D.H. and King, W.A. 2006. Role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer. BMC Dev Biol 6(1):41. https:// doi.org/10.1 186/1471-213X-6-41 Martins, C., Fonseca, I., Felix, A., Roque, L. and Soares, J. 1995. Benign salivary gland tumors: A cytogenetic study of 21 cases. J Surg Oncol 60(4):232-237. https://doi. org/10.1002/jso.2930600404 Matse J.H., Veerman E.C.I., Bolscher J.G.M., René Lee-mans C., Ylstra B. and Bloemena E. 2017. High number of chromosomal copy number aberrations inversely relates to t(11;19)(q21;p13) translocation status in muco-epidermoid carcinoma of the salivary glands. Oncotarget 8(41): 69456-69464. https://doi.org/10.18632/oncotar-get.17282
Murase, R., Sumida, T., Kawamura, R., Onishi-Ishikawa, A., Hamakawa, H., McAllister, S.D. and Desprez, P. 2016. Suppression of invasion and metastasis in aggressive salivary cancer cells through targeted inhibition of ID1 gene expression. Cancer Lett 377(1):11-16. https://doi. org/10.1016/j.canlet.2016.04.021 Müller, S. 2013. An update on salivary gland pathology. Head Neck Pathol 7 (Suppl 1):S1-S2. https://doi.org/10.1007/ s12105-013-0463-y
Nielsen, L.L., Gurnani, M., Porter, G., Trexler, S., Emerson, D. and Tyler, R.D. 1994. Development of a nude mouse model of ras-mediated neoplasia using WR21 cells from a transgenic mouse salivary tumor. In Vivo 8(3):295-302.
Omitola, O.G. and Iyogun, C.A. 2018. Immunohistochemi-cal study of salivary gland tumors in a tertiary institution in South-South Region of Nigeria. J Oral Maxillofac Pathol 22(2):163-167. https://doi.org/10.4103/jomfp. JOMFP_108_17
Palanisamy, V., Park, N.J., Wang, J. and Wong, D.T. 2008. AUF1 and HuR proteins stabilize interleukin-8 mRNA in human saliva. J Dent Res 87(8):772-776. https://doi. org/10.1177/154405910808700803 Persson, F., Andrén, Y., Winnes, M., Wedell, B., Nordkvist, A., Gudnadottir, G., Dahlenfors, R., Sjögren, H., Mark, J. and Stenman, G. 2009. High-resolution genomic profiling of adenomas and carcinomas of the salivary glands reveals amplification, rearrangement, and fusion of HMGA2. Genes Chromosomes Cancer 48(1):69-82. https://doi. org/10.1002/gcc.20619 Rao, P.H., Roberts, D., Zhao, Y., Bell, D., Harris, C.P., Weber, R. S. and El-Naggar, A. K. 2008. Deletion of 1p32-p36 is the most frequent genetic change and poor prognostic marker in adenoid cystic carcinoma of the salivary glands. Clin Cancer Res 14(16):5181-5187. https://doi. org/10.1158/1078-0432.CCR-08-0158 Rhode, H., Liehr, T., Kosyakova, N., Rincic, M. and Aza-wi, S. S. H. 2018. Molecular cytogenetic characterization of two murine colorectal cancer cell lines. OBM Genetics 2(3). https://doi.org/10.21926/obm.genet.1803037 Seethala, R.R. 2017. Salivary gland tumors: current concepts and controversies. Surg Pathol Clin 10(1):155-176. https://doi.org/10.1016Zj.path.2016.11.004 Solanki, G. 2011. Tumors of salivary glands. IJPR 1(2):35-38.
https://doi.org/10.7439/ijpr.v1i2.355 Sood, S., McGurk, M. and Vaz, F. 2016. Management of salivary gland tumours: United Kingdom national multidis-ciplinary guidelines. J Laryngol Otol 130(S2):S142-S149. https://doi.org/10.1017/S0022215116000566 Trzaskawka, E., Vigo, J., Egea, J.-C., Goldsmith, M.-C., Salmon, J.-M., Deville and De Periere, D. 2000. Cultured tumor cells of murine submandibular gland origin: a model to investigate pHi regulation of salivary cells. Eur J Oral Sci 108(1):54-58. https://doi.org/10.1034Zj.1600-0722.2000.00670.x Vekony, H., Röser, K., Löning, T., Ylstra, B., Meijer, G.A., van Wieringen, W. N., van de Wiel, M.A., Carvalho, B., Kok, K., Leemans, S. R., van der Waal, I. and Bloemena, E. 2009. Copy number gain at 8q12.1-q22.1 is associated with a malignant tumor phenotype in salivary gland myoepi-theliomas. Genes Chromosomes Cancer 48(2):202-212. https://doi.org/10.1002/gcc.20631 Yin, L.X. and Ha, P.K. 2016. Genetic alterations in salivary gland cancers. Cancer 122(12):1822-1831. https://doi. org/10.1002/cncr.29890 Young, L. F. and Martin, K. R. 2006. Time-dependent resvera-trol-mediated mRNA and protein expression associated with cell cycle in WR-21 cells containing mutated human c-Ha-Ras. Mol Nutr Food Res 50(1):70-77. https://doi. org/10.1002/mnfr.200500149 Zboray, K., Mohrherr, J., Stiedl, P., Pranz, K., Wandruszka, L., Grabner, B., Eferl, R., Moriggl, R., Stoiber, D., Sakamoto, K., Wagner, K., Popper, H., Casanova, E. and Moll, H. 2018. AKT3 drives adenoid cystic carcinoma development in salivary glands. Cancer Med 7(2):445-453. https://doi.org/10.1002/cam4.1293
Supplement 1. Translation of murine to human data for WR21
region gain homologue region in human
cytoband position (GRCh37/hg19)
3D-E3 X1 3q25.1-q26.2 3:149055816-167822106
3C X1 4q27-q31.1 4:122242382-141190230
8A1-E2 X1 19p13.2 13q33.1-q34 8p23.3-p23.2 8p23.2-p23.1 gap 13q14.3 8p11.23-p11.21 8p11.21 8p12 8p23.1 8p23.1-p22 4q32.2-q35.2 8p22-p21.3 19p13.12-p13.11 22q12.3 4q31.1-q31.23 19p13.2-p13.12 16q11.2-q22.1 16q22.1-q24.3 1q42.13-q42.3 10p11.22-p11.21 19:7112183-8071013 13:103533915-115092930 8:591286-5358752 8:5368147-6693649 13:52435459-53211718 8:36716542-42505949 8:42691750-43058925 8:29190466-36677574 8:8108776-9640417 8:12579073-17958954 4:163504024-190884657 8:18227877-20177976 19:16163040-19774937 22:33658332-35953121 4:141251922-150892329 19:12745060-14683008 16:46693273-69976105 16:70109527-90110030 1:229404294-235324774 10:33112469-35152269
9E1-E2 X1 6q13-q14.3 6:74104388-86360515
12F1-F2 X1 No discerption (Gap) No discerption (Gap)
19A-D X1 11q12.1-q13.3 9q21.11-q21.31 2q13 9p24.3-p24.1 10q11.23-q21.1 11:57844834-68709722 9:69086307-82777364 2:114171139-114321953 9:51374-6659223 10:51917603-54540082
17E5 X1 18p11.32 18:861722-2534400
region loss homologue region in human
cytoband position (GRCh37/hg19)
2C3 X1 2q22.1-q32.1 2:139292421-187530602
region breakpoint homologue region in human
cytoband potential tumor associated genes
4C4 inv. 9p22.3~22.2 PSIP1
8A1 t 19p13.2 ADGRE4P
13D2 add 5p12 FGF10
Supplement 2. Translation of murine to human data for SCA-9
region gain homologue region in human
cytoband position (GRCh37/hg19)
4A5-B3 X1 9q22.33-q33.2 9:100037894-123488942
9p13.1-p21.2 9:27325073-38472099
5G2-G3 X2 13q12.13-q13.2 13:26784894-34260463
7q21.1-q21.3 7:97598308-99229367
6A1-qter x1 7p21.3-p22.1 7:7132996-12536829
7q21.2-q21.3 7:92745197-97502117
12p11.21 12:30985917-31165338
12p11.21 12:31424829-32537434
12p11.22-p13.31 12:9901365-30943693
12p13.31- p13.33 12:2903120-7695890
12p13.31 12:8071763-9214464
12p13.33 12:66113-2823666
10q11.21-q11.22 10:43277986-46218167
4q27 4:121018693-122194687
4q22.1-q22.3 4:89178698-95273100
3p25.2-ptr 3:61304-12897767
3p12.3-p14.1 3:64017713-75322601
3q21.3 3:125725101-129038484
3p25.1-p25.2 3:12939278-15163105
3q21.3-q22.1 3:129094932-129632650
2p11.2-p13.3 2:68715037-87095119
2p11.2 2:88302422-89174373
1p31.1 1:67631910-68317098
7p14.3-p15.3 7:23254035-33103246
7q31.1-q36.1 7:112138919-149583263
7q36.1 7:150032467-150558657
22q11.11-q11.21 22:17565811-18659740
8A4-E2 X1 4q32.2-qtr 4:163504024-190884657
4q31.1-q31.23 4:141251922-150892329
16q22.1-q24.3 16:70109527-90110030
16q11.2-q22.1 16:46693273-69976105
1q42.13-q42.3 1:229404294-235324774
10q11.21-q11.22 10:33112469-35152269
19p13.11-p13.12 19:16163040-19774937
19p13.12-p13.2 19:12745060-14683008
22q12.3 22:33658332-35953121
8p22-23.1 8:12579073-17958954
homologue region in human
region
loss
cytoband position (GRCh37/hg19)
1A2-H6 x1 8q11.21-q12.1 8:50767106-56535248
8q13.1-q21.11 8:67336477-76107163
6p12.3-p12.2 6:49796129-52568703
6q11-q13 6:61967179-73920868
6p12.1-p11.2 6:56223874-58686221
2q14.3-q21.1 2:128848553-131914911
2q11.2-q12.2 2:97151065-106819719
13q33.1 13:103237605-103533914
2q32.1-q32.2 2:189007277-190504466
2q32.2-q37.3 2:190506076-242812118
5q21.1 5:98439740-102728411
18q21.32-q22.1 18:58351903-65328593
2q14.3 2:122585948-126347698
2q14.1-q14.3 2:114436107-122578025
2q21.2-q22.1 2:133138389-138607743
1q32.1-q32.2 1:206075775-207534964
1q21.1 1:143881371-144095755
1p11.2 1:120754434-120887322
1q23.1-q32.1 1:158516903-205922697
Yq11.23 Y:28358518-28544030
1q43-q44 1:240253393-247125743
4q26 4:119339188-119512723
1q32.2-q42.13 1:207575939-227644727
2A1-A3 X1 10q12.1-q15 10:5915452-27157072
2B X4 2q22.1-q32.1 2:140065297-188395329
2C1-H4 X1 20p13-p11.21 20:1736101-25606620
20p13 20:102147-1447942
20q11.21-q13.32 20:29933153-58056214
20q13.32-q13.33 20:58148222-62907435
15q13.3-q21.2 15:32906987-51298173
11q12.1 11:56082416-57753858
11q11 11:55080583-55323018
11p11.12 11:51377850-51539057
11p14.2-p11.2 11:26296397-48658712
2q11.1-q11.2 2:95642277-97040617
2q13 2:111483204-112960231
2p11.2 2:87345633-87996071
2q13 2:112973390-113650007
4C5-E1 X1 1p32.1-p31.3 1:59120351-67562260
1p36.33-p32.2~1 1:894315-59012766
7A1-F3 X1 19q13.42-q13.43 19:54368915-57485284
19q13.43 19:58523795-59089552
19q13.31-q13.33 19:45010010-48707700
19q12-q13.31 19:30093064-44860951
19q12 19:28589680-30085362
19q13.33-q13.41 19:48800017-51921957
16p13.11 16:16252815-16388674
11p15.1-p14.3 11:17403485-25251145
15q11.2 15:22833222-23086601
15q11.2-q13.1 15:23914751-28586067
15q13.1-q13.3 15:29107424-32578594
15q26.3 15:99080385-102265870
15q26.1-q26.3 15:91593058-99078056
15q25.3-q26.1 15:85829657-91565912
15q25.1-q25.3 15:80253398-85682414
11p11.12 11:49250334-49827246
11q13.4-q14.3 11:71627032-89350901
10p11.21 10:37191655-37402201
11p15.4-p15.1 11:3631069-17360027
16p13.11 16:15260325-15369270
16p13.11-p12.3 16:16681590-18325190
16p12.3-p12.2 16:18608156-21351663
16p12.2-p11.2 16:21572755-28339524
16p11.2 16:28390845-29030948
16p11.2 16:29661006-31520748
9A1-F4 11q14.3-q22.3 11:89860533-107436639
19p13.2 19:8919008-11689880
7p14.3-p14.2 7:33134362-36494039
11q22.3-q25 11:107452617-134843539
15q21.2 15:51349622-51942502
15q21.2-q25.1 15:51961808-78956872
6p12.2-p12.1 6:52656530-55784577
6q13-q14.3 6:74104388-86360515
15q25.1 15:79042978-80196839
3q22.3-q24 3:138372654-148087492
3q22.1-q22.3 3:129931635-138353358
3p21.31-p21.1 3:46446256-52346387
3p24.1-p22.2 3:27753690-37261140
3p22.2-p21.31 3:37269243-46423369
12A1-E X2 2p25.1-p23.3 2:10303009-26361943
2p25.1 2:9354723-9994801
2p25.1 2:9996101-10284917
2p25.3-p25.1 2:140908-9278318
7q22.3-q31.1 7:105210238-107772185
7p21.3-p21.1 7:12561752-19748810
7q31.1 7:107772206-112136146
14q12-q22.1 14:25157192-52251174
12E-F1 X1 14q23.1-q32.33 14:58666612-106375879
12F1-F2 X2 7q36.3 7:157225645-158937901
7p21.1-p15.3 7:19761201-22528893
13A1-qter X1 10p15.3-p15.1 10:138698-5865622
1q42.3-q43 1:235330060-240084659
7p14.2-p13 7:36524506-43605930
6p22.3-p22.1 6:20065223-28502803
6p25.3-p23 6:181261-15099150
6p23-p22.3 6:15104709-20060798
9q22.1-q22.32 9:91031851-97067712
5q35.2-q35.3 5:173750964-177039611
5q31.1-q31.2 5:134073478-137090938
9q21.32-q21.33 9:86231955-90340399
9q22.32-q22.33 9:97320957-99417669
9p13.1 9:38810965-40707569
9q12-q13 9:65585614-65901647
9p11.2 9:43623473-43941731
8q22.1 8:97247028-97373828
5p15.33-p15.31 5:191425-7935441
5q14.3-q15 5:84566270-96144383
5q13.2-q14.3 5:70265557-84371909
5q11.1-q13.2 5:49569996-68922426
1p11.2 1:121149401-121350677
5p12 5:43446298-46118514
14C1-E5 X2 14q22.1-q23.1 14:52688635-58629894
14q11.2-q12 14:20211286-24987352
14q12 14:25040539-25149959
13q12.12 13:25188452-25511922
13q12.11 13:20207279-23370461
13q14.2 13:49821990-50161404
13q12.13 13:25685086-26668986
13q12.12 13:23853398-24896355
13q14.2-q14.3 13:50192169-52356487
8p23.1 8:9744629-11737304
8p21.3-p12 8:20206584-29151199
13q14.11-q14.2 13:41469941-49799059
13q14.3-q33.1 13:53226033-103089581
16pter-qter X1 16p13.3-p13.11 16:3283710-15197331
16p13.11 16:15478874-16187414
8q11.21 8:48206338-49865275
12p11.21 12:32634919-33054761
22q11.21 22:19010381-22338262
3q27.1-q29 3:182965714-195325931
3q29 3:195428230-197771581
3q11.1-q21.2 3:93527487-125343459
3p12.3-p11.1 3:75865702-90309600
21q11.2-q22.3 21:15515528-43438088
21q11.2 21:14535253-14714360
18p11.21 18:15016525-15155234
2q21.1 2:132604281-132757591
17A1-E5 X1 6q27 6:167120855-167552070
6q25.3-q27 6:160103032-166797236
6q27 6:167859539-170893754
5q15-q21.1 5:96202316-98405239
16p13.3 16:222880-3208490
5q35.1 5:171946752-172722349
6p21.32-p21.2 6:33359177-39058058
21q22.3 21:43490502-45122943
19p13.12 19:15270296-15808207
19p13.2 19:8366687-8811037
6p22.1-p21.32 6:29322703-33297218
6p21.2-p12.3 6:39266498-49681826
3p25.1-p24.3 3:16307846-20231899
2q12.2-q12.3 2:107383985-108798215
19p13.3 19:4229082-6862967
5q21.1-q22.1 5:102759315-110063021
18p11.32-p11.22 18:2534401-9972541
2p23.2-p16.3 2:29033520-51699597
2p16.3-p16.2 2:51709987-53282184
18p11.32 18:861722-2534400
18A1-D3 X1 10p11.21 10p12.1-p11.22 10p12.1 10p11.21 18p11.32 18q11.1-q12.3 2q14.3 5q22.1-q22.2 5q31.2-q32 5q22.2-q23.3 10:35284099-35521818 10:28950711-32678701 10:27747786-28722506 10:35676708-37094546 18:112543-599224 18:18528605-41073893 2:127805408-128786667 5:110280120-112296881 5:137225085-147624774 5:112310736-130339352
18D3-qter X2 5q32-q33.1 18p11.22-p11.21 18q21.31-q21.32 18p11.21 18q12.3-q21.31 18q22.1-q23 5:147647374-150177176 18:10202644-11518916 18:54267924-58201586 18:11649353-13871680 18:41355914-54244819 18:66339761-78010601
region breakpoint homologue region in human
cytoband potential tumor associated genes
4C1 del 9q31.3 AKAP2/ C9orf84
5G3 dup, inv 7q21.3 ASNS / BAIAP2L1
5G2 dup 7q21.3 ASNS / BAIAP2L1
8A1 t 19p13.2 ELAVL1 also called HUR / FCER2
8B1 t 4q34.2 ASB5
12E t 14q31.1 SEL1L / TSHR
14C1 t 14q22.2 CGRRF1 / CNIH1 / GCH1 / GMFB
18D t 5q23.1 LOX / FTMT
