Increase, Proliferation of goblet cells

Cell proliferation is defined as the increase in cell number because of cell growth and division (Yada et al., 2014). It is a tightly regulated process. In adult organisms, cells are normally in a non-proliferative state, and cell proliferation primarily occurs to replace cells lost due to cell death. Because of tissue injury or exposure to chemicals, cell proliferation may take place at an accelerated pace, skewing the balance between cell growth and division and cell death.

Goblet cells proliferate under normal conditions (Chopra et al., 1981) and in response to a variety of stimulants including EGF, viruses, bacteria and allergens (Ichinose et al., 2006; Camateros et al., 2007; Shatos et al., 2003; Duh et al., 2000; Tamaoki et al., 2004) in colon, eye, nose and lung. EGFR ligands EGF, HB-EGF and TGFA induce goblet cell proliferation through MAPK, p38 and JNK in cultured conjunctival and bronchial goblet cells (Booth et al., 2001; Booth et al., 2007; Gu et al., 2008; Shatos et al., 2003).

Various methods for measuring cell proliferation exist. A number of recent reviews summarize the available methods, their principles, advantages and drawbacks (Riss et al., 2013; Cobb, 2013; Yadav et al., 2014; Adan et al., 2016; Romar et al., 2016). Broadly, cell proliferation assays can be categorized on the basis of their principle, and the 3 major categories are based on measurments of: 1) rate of DNA synthesis, 2) metabolic activity of cells, and 3) antigens associated with cell proliferation.

Assays that measure the rate of DNA synthesis

During cell proliferation, DNA replication occurs before cell division starts; therefore, the rate of DNA replication/synthesis is directly proportional to the rate of cell proliferation (Yadav et al., 2014). If cell proliferation is negatively affected by exposure, nascent DNA content would be lower in treated than in untreated cells. Conversely, if a chemical increases proliferation, the rate of DNA synthesis would increase.

Incubation of proliferating cells with labeled nuclotides such as ³H-thymidine and other thymidine analogs such as BrdU leads to their incorporation in newly synthesized DNA. Scintigraphy can then be used to detect the radiolabeled tracer and quantify the amount incorporated. BrdU can be detected by using anti-BrdU antibody-based staining and ELISA or flow cytometry or, if labeled with a fluorochrome, directly by reading fluorescent signals with a plate reader. Because directly labeled BrdU can also be detected microscopically, high-content imaging may prove to be another suitable alternative.

Because assays in this category are sensitive to cell cycle phase, synchronization of cells either by serum withdrawal (which accumulates cells in G1) or chemical inhibition of DNA synthesis (blocking cells in S phase) should be considered.

Assays that measure metabolic activity

Increased metabolic activity is typical for actively proliferating cells and is characterized by increasing ratios of NADPH/NADP, FADH/FAD, FMNH/FMN, and NADH/NAD. The oxidant capabilities of these metabolic intermediates cellular dehydrogenases or reductases can be exploited to convert e.g. tetrazolium salts to a formazan product, resulting in a colorimetric change which can be detected by measuring absorbance at an appropriate wavelength with a plate reader. There are several tetrazolium salts, including MTT, MTS/XTT, and WST, that can be used for this purpose. They are also included in various commercially available kits.

Other dyes that are sensitive to changes in metabolic activity, such as resazurin can also be used to determine the number of viable cells. Similar to the tetrazolium salts, resazurin is reduced in viable cells, giving rise to resorufin and dihydroresorufin, which have a different absorbance than resazurin and can be measured by using a plate reader. In addition, ATP concentration can serve as an indicator of cell number, because ATP levels are rapidly depleted and not restored through new synthesis when cell death occurs. Thus, ATP content directly correlates with the rate of proliferation of cells. Commonly, bio- or chemiluminescent assays, most of which are commercially available, are used for measuring ATP content.

Assays that assess antigens associated with cell proliferation

Proliferating cells express key proteins or antigens that are not present in non-proliferating cells, such as proliferating cell nuclear antigen (PCNA), Ki-67, topoisomerase IIB and phospho-histone H3. These can be detected with appropriate antibodies and imaging techniques. Immunocytochemistry/immunohistochemistry are commonly applied to verify proliferation in tissue sections, and here, tissue context may add insights into specific subpopulations of cells that stain positively for these markers. Semiquantitative image analysis may provide means to quantify a proliferation index. Flow cytometry and high-content imaging can be applied to cell cultures or ex vivo cell samples (e.g. PBMC). Although no standardized methods for the detection of these antigens exist, there are several well-described protocols for some of these markers for clinical application (Padmanabhan, 2015; Sun et al., 2016; Winther et al., 2016; Shen et al., 2017; Volynskaya et al., 2019; Nielsen et al., 2020)

It is worth noting here that at least one OECD Test Guideline (TG) refers to cell proliferation assays as essential components of the assessment of chemicals in vitro. Specifically, TG No. 487 (In Vitro Mammalian Cell Micronucleus Test) recommends the measurement of Relative Population Doubling (RPD) or Relative Increase in Cell Count (RICC) and Proliferation Index (PI) (OECD, 2016).

There are also a number of recent developments that changed the manner in which cell proliferation can be assessed today. For example, real-time cell analysis (RTCA) is based on electrical impedance measurements and allows for label-free, real-time, and continuous monitoring of cell adhesion, morphology, and rate of cell proliferation (Yan et al., 2018; Stefanowicz-Hajduk and Ochocka, 2020). 

Proliferation of goblet cells was reported in human, mouse and rat (Tamaoki et al., 2004; Ichinose et al., 2006; Camateros et al., 2007; Shatos et al., 2003; Duh et al., 2000; Sydlik et al., 2006; Taniguchi et al., 2011).

Booth, B.W., Sandifer, T., Martin, E.L., and Martin, L.D. (2007). IL-13-induced proliferation of airway epithelial cells: mediation by intracellular growth factor mobilization and ADAM17. Respir. Res. 8, 51.

Camateros, P., Tamaoka, M., Hassan, M., Marino, R., Moisan, J., Marion, D., Guiot, M.-C., Martin, J.G., and Radzioch, D. (2007). Chronic asthma-induced airway remodeling is prevented by toll-like receptor-7/8 ligand S28463. Am. J. Respir. Crit. Care Med. 175, 1241–1249.

Chopra, D.P., Yeh, K., and Brockman, R.W. (1981). Isolation and characterization of epithelial cell types from the normal rat colon. Cancer Res. 41, 168–175.

Cobb, L. (2013). Cell Proliferation Assays and Cell Viability Assays. MATER METHODS 3, 2799.

Duh, G., Mouri, N., Warburton, D., and Thomas, D.W. (2000). EGF regulates early embryonic mouse gut development in chemically defined organ culture. Pediatr. Res. 48, 794–802.

Gu, J., Chen, L., Shatos, M.A., Rios, J.D., Gulati, A., Hodges, R.R., and Dartt, D.A. (2008). Presence of EGF growth factor ligands and their effects on cultured rat conjunctival goblet cell proliferation. Exp. Eye Res. 86, 322–334.

Haswell, L.E., Hewitt, K., Thorne, D., Richter, A., and Gaça, M.D. (2010). Cigarette smoke total particulate matter increases mucous secreting cell numbers in vitro: A potential model of goblet cell hyperplasia. Toxicol. in Vitro 24, 981-987. 

Haswell, L.E., Smart, D., Jaunky, T., Baxter, A., Santopietro, S., Meredith, S., et al. (2021). The development of an in vitro 3D model of goblet cell hyperplasia using MUC5AC expression and repeated whole aerosol exposures. Toxicol. Lett. 347, 45-57. 

Ichinose, T., Sadakane, K., Takano, H., Yanagisawa, R., Nishikawa, M., Mori, I., Kawazato, H., Yasuda, A., Hiyoshi, K., and Shibamoto, T. (2006). Enhancement of mite allergen-induced eosinophil infiltration in the murine airway and local cytokine/chemokine expression by Asian sand dust. J. Toxicol. Environ. Health A 69, 1571–1585.

Shatos, M.A., Ríos, J.D., Horikawa, Y., Hodges, R.R., Chang, E.L., Bernardino, C.R., Rubin, P.A.D., and Dartt, D.A. (2003). Isolation and characterization of cultured human conjunctival goblet cells. Invest. Ophthalmol. Vis. Sci. 44, 2477–2486.

Shen, Y., Vignali, P., and Wang, R. (2017). Rapid Profiling Cell Cycle by Flow Cytometry Using Concurrent Staining of DNA and Mitotic Markers. Bio Protoc. 7, e2517.

Sun, Y., Yang, K., Bridal, T., and Ehrhardt, A.G. (2016). Robust Ki67 detection in human blood by flow cytometry for clinical studies. Bioanalysis 8, 2399-2413. 

Sydlik, U., Bierhals, K., Soufi, M., Abel, J., Schins, R.P.F., and Unfried, K. (2006). Ultrafine carbon particles induce apoptosis and proliferation in rat lung epithelial cells via specific signaling pathways both using EGF-R. Am. J. Physiol. Lung Cell. Mol. Physiol. 291, L725–L733.

Tamaoki, J., Isono, K., Takeyama, K., Tagaya, E., Nakata, J., and Nagai, A. (2004). Ultrafine carbon black particles stimulate proliferation of human airway epithelium via EGF receptor-mediated signaling pathway. Am. J. Physiol. Lung Cell. Mol. Physiol. 287, L1127–L1133.

Taniguchi, K., Yamamoto, S., Aoki, S., Toda, S., Izuhara, K., and Hamasaki, Y. (2011). Epigen is induced during the interleukin-13-stimulated cell proliferation in murine primary airway epithelial cells. Exp. Lung Res. 37, 461–470.

Volynskaya, Z., Mete, O., Pakbaz, S., Al-Ghamdi, D., and Asa, S.L. (2019). Ki67 Quantitative Interpretation: Insights using Image Analysis. J. Pathol. Inform. 10, 8.

Winther, T.L., Arnli, M.B., Salvesen, Ø., and Torp, S.H. (2016). Phosphohistone-H3 Proliferation Index Is Superior to Mitotic Index and MIB-1 Expression as a Predictor of Recurrence in Human Meningiomas. Am. J. Clin. Pathol. 146, 510-520. 

Yadav K, Singhal N, Rishi V, Yadav H (2014). Cell Proliferation Assays. In: eLS. John Wiley & Sons, Ltd: Chichester. 

Yan, G., Du, Q., Wei, X., Miozzi, J., Kang, C., Wang, J., et al. (2018). Application of Real-Time Cell Electronic Analysis System in Modern Pharmaceutical Evaluation and Analysis. Molecules 23, 3280.