Retinoblastoma is an intraocular cancer occurring in early childhood, caused by a disruption in the tumor suppressor RB gene. [1] The Y79 and WERI-RB27 cell lines are established retinoblastoma cell lines containing deletions in the RB gene. [2] [3] [4] [5]

There is considerable debate as to the origin of retinoblastoma cells. One hypothesis implicates cancer stem cells, which are thought to cause chemotherapy resistance. We previously documented the expression of stem cell markers in retinoblastoma, providing evidence in support of the stem cell hypothesis. [6] [7]

In this report, we sought to compare the expression of mature retinal markers MAP2, PKCalpha, CRX, recoverin and Tuj1 (beta tubulin) in commonly studied retinoblastoma cell lines Y79 and WERI-RB27. We tested the hypothesis that these two cell lines would differentially express mature retinal markers, in addition to the previously reported stem cell markers.

Cell culture
The retinoblastoma cell lines Y79 and WERI-RB27 were grown in suspension in Dulbecco's Modified Eagle Medium (DMEM) with 10% calf serum (Life Technologies, Grand Island, NY) in a 37°C incubator with 5% CO2. MDA-MB-231, a human breast adenocarcinoma cell line, was grown as a control in Leibovitz's L15 medium (ATCC, Manassas, VA) with 10% calf serum at 37°C in 100% room air.

Immunocytochemistry
Immunostaining was performed as previously described. [6] [7] Cells were harvested, resuspended in cytospin solution (72% isopropanol, 19% acetone, 7.6% glycerol) and were spun onto slides using a Shandon Cytospin II. Slides were blocked with enzyme/protein block (Abcam, Cambridge, MA), followed by one hour incubation in primary antibody at the appropriate concentration (Table 1) or isotype control antibody. Slides were washed with PBS and incubated in secondary polymer (Syd Laboratories, Malden, MA). Staining with DAB (diaminobenzidine) was performed for five minutes, and the slides were washed with water before being covered with cover slip. Three groups of 100 cells were counted as immunopositive for each slide. Slides were photographed using a SONY ICX 285AL SPOT camera (Diagnostic Instruments, Sterling Heights, MI). Each experiment was repeated three times and graphed as shown in Figure 1.

q RT PCR of mature retinal markers
RNA from Y79 and WERI-RB27 cells was prepared using the RNeasy Plus kit (Qiagen, Valencia, CA). One µg of RNA was reverse transcribed using the iScript cDNA synthesis kit (BioRad, Hercules, CA). qPCR was performed using Sybr Green Master mix (Qiagen) and the primers listed in Table 2 (IDT, Coralville, IA). The reactions were run in a BioRad My iQ cycler using the following conditions: MAP2, Tuj1: 40 cycles; 95°C 15 sec, 60°C 1 min. CRX, recoverin, PKCa: 35 cycles; 95°C 30 sec, 60°C 30 sec, 72°C 30 sec.

Statistical analysis
The percentage of immunoreactive cells for three repeated experiments for each marker was graphed using Prism software (Graphpad, La Jolla, CA). For qPCR analysis, relative expression was calculated with the 2-delta, delta Ct equation using GAPDH as the reference gene. Three repeats of each experiment were performed using different cell preparations and the results were graphed using Prism software (Graphpad, La Jolla, CA).

Retinoblastoma cells express mature markers at the protein level
Cells were tested for CRX, Tuj1, recoverin, MAP2 and PKC alpha expression by immunocytochemsitry. WERI-RB27 showed high levels of recoverin and MAP2 expression with 35% and 25% of cells staining positive respectively. Two and 10% of Y79 cells were immunopositive for recoverin and MAP2 respectively (Figure 1). CRX, Tuj-1 and PKCa showed similar levels of protein expression between the two cell lines, with 5–10% of the cells being immunoreactive. Controls were immunonegative

Retinoblastoma cells express mature markers at the RNA level
For qPCR analysis, mature retinal marker expression was compared in Y79, WERI-RB27 and MDA-MB231, a breast cancer control cell line. Y79 expression was set at one and the other cell types were compared to it. MDA-MB231 cells exhibited low levels of all mature retinal markers except for PKCa, as expected, as these cells are known to contain high levels of PKCa [8]. Relative levels of recoverin expression were similar in both cell lines using qPCR. RNA expression levels were about two times higher in Y79 for PKCa, Tuj-1, and CRX. MAP2 RNA expression was 7–10 times higher in WERI-RB27 cells than in Y79 cells (Figure 2).

We previously noted the presence of stem cell markers in retinoblastoma cell lines. [6] [7] Here we report on the comparative levels of mature retinal markers in these cells. Y79 and WERI-RB27 cell lines both exhibit expression of mature markers CRX, PKCa, recoverin, Tuj1 and MAP2 at the protein and mRNA levels.

The origin of retinoblastoma is controversial, with some evidence implying a neuronal lineage, as indicated by expression of neuronal markers MAP2 (Microtubule-associated protein- 2) and beta tubulin. [9] [10] [11] [12] The data regarding MAP2 expression in the present study corroborate this conclusion, as MAP2 was found to be expressed at high levels in WERI-RB27 at both the protein and mRNA levels.

Recoverin is a photoreceptor marker [13] [14] and was found to be present in high levels in WERI-RB27 using immunocytochemistry. The discrepancy between protein and RNA expression of recoverin may be explained by post-transcriptional processing.

Lower levels of CRX, a photoreceptor homeobox transcription factor, [15] [16] [17] and Tuj1, a retinal ganglion cell marker, [18] were expressed by both cell types at the protein and mRNA levels.

MDA-MB231 cells expressed high levels of PKCa, a bipolar cell marker in the retina, [19] [20] as the marker also labels aggressive breast cancer cells. [8]

WERI-RB27 and Y79 retinoblastoma cell lines express a variety of mature markers representing several retinal cell types. This observation reinforces the potential capacity for differentiation into more mature cells. Future studies into the nature of these cells are warranted, as these markers may potentially be used as target endpoints for differentiation therapies.

This work was supported by the Cornell Center on the Microenvironment and Metastasis through Award Number U54CA143876 from the National Cancer Institute, R21CA127061 and NYSTEM C026412. We thank Dr. Catherine Foster and Dr. Senthil Manohar for technical advice and assistance.

1. Viatour P, Sage J. Newly identified aspects of tumor suppression by RB. Dis Model Mech 2011;4(5):581–5.   [CrossRef]   [Pubmed]    
2. Reid TW, Albert DM, Rabson AS, et al. Characteristics of an established cell line of retinoblastoma. J Natl Cancer Inst 1974;53(2):347–60.   [Pubmed]    
3. Sery TW, Lee EY, Lee WH, et al. Characteristics of two new retinoblastoma cell lines: WERI-Rb24 and WERI-Rb27. J Pediatr Ophthalmol Strabismus 1990;27(4):212–7.   [Pubmed]    
4. Madreperla SA, Bookstein R, Jones OW, Lee WH. Retinoblastoma cell lines Y79, RB355 and WERI-Rb27 are genetically related. Ophthalmic Paediatr Genet 1991;12(1):49–56.   [CrossRef]   [Pubmed]    
5. Lee EY, Bookstein R, Young LJ, Lin CJ, Rosenfeld MG, Lee WH. Molecular mechanism of retinoblastoma gene inactivation in retinoblastoma cell line Y79. Proc Natl Acad Sci U S A 1988;85(16):6017–21.   [CrossRef]   [Pubmed]    
6. Seigel GM, Campbell LM, Narayan M, Gonzalez-Fernandez F. Cancer stem cell characteristics in retinoblastoma. Mol Vis 2005;11:729–37.   [Pubmed]    
7. Seigel GM, Hackam AS, Ganguly A, Mandell LM, Gonzalez-Fernandez F. Human embryonic and neuronal stem cell markers in retinoblastoma. Mol Vision 2007;13:823–2.   [Pubmed]    
8. Lønne GK, Cornmark L, Zahirovic IO, Landberg G, Jirström K, Larsson C. PKCalpha expression is a marker for breast cancer aggressiveness. Mol Cancer 2010;14;9:76.   [Pubmed]    
9. Sakata R, Yanagi Y. Expression of immature and mature retinal cell markers in retinoblastoma. Eye 2008;22(5):678–3.   [CrossRef]   [Pubmed]    
10. Gass P, Frankfurter A, Katsetos CD, Herman MM, Donoso LA, Rubistein LJ. Antigenic expression of neuron-associated class III beta-tubulin isotype (h beta 4) and microtubule-associated protein 2 (MAP2) by the human retinoblastoma cell line WERI-Rb1. A comparative immunoblot and immunocytochemical study. Opthalmic Res 1990;22(1):57–66.   [Pubmed]    
11. Herman MM, Perentes EE, Katseos CD, et al. Neuroblastic differentiation potential of the human retinoblastoma cell lines Y-79 and WERI-Rb1 maintained in an organ culture system. An immunohistochemical, electron microscopic, and biochemical study. Am Journal of Pathology 1989;134(1):115–32.   [Pubmed]    
12. Katsetos CD, Herman MM, Frankfurter A, Uffer S, Perentes E, Rubinstein LJ. Neuron-associated class III beta-tubulin isotype, microtubule-associated protein 2, and synaptophysin in human retinoblastomas in situ. Further immunohistochemical observations on the Flexner-Wintersteiner rosettes. Lab Invest 1991;64(1):45–4.   [Pubmed]    
13. Wiechmann AF. Recoverin in cultured human retinoblastoma cells: enhanced expression during morphological differentiation. J Neurochem 1996;67(1):105–10.   [Pubmed]    
14. Dizhoor AM, Ray S, Kumar S, et al. Recoverin: a calcium sensitive activator of retinal rod guanylate cyclase. Science 1991 Feb 22;251(4996):915–8.   [CrossRef]   [Pubmed]    
15. Santagata S, Maire CL, Idbaih A, et al. CRX is a diagnostic marker of retinal and pineal lineage tumors. PLoS One 2009 Nov 20;4(11):e7932.   [CrossRef]   [Pubmed]    
16. Glubrecht DD, Kim JH, Russell L, Bamforth JS, Godbout R. Differential CRX and OTX2 expression in human retina and retinoblastoma. J Neurochem 2009 Oct;111(1):250–63.   [CrossRef]   [Pubmed]    
17. Swaroop A, Kim D, Forrest D. Transcriptional regulation of photoreceptor development and homeostasis in the mammalian retina. Nat Rev Neurosci 2010 Aug;11(8):563–76.   [CrossRef]   [Pubmed]    
18. Zhang XM, Li Liu DT, Chiang SW, et al. Immunopanning purification and long-term culture of human retinal ganglion cells. Mol Vis 2010 Dec 28;16:2867–2.   [Pubmed]    
19. Greferath U, Grünert U, Wässle H. Rod bipolar cells in the mammalian retina show protein kinase C-like immunoreactivity. J Comp Neurol 1990 Nov 15;301(3):433–2.   [CrossRef]   [Pubmed]    
20. Zhang DR, Yeh HH. Protein kinase C-like immunoreactivity in rod bipolar cells of the rat retina: a developmental study. Vis Neurosci 1991 May;6(5):429–37.   [CrossRef]   [Pubmed]