Expression of alpha-crystallin in retinoblastoma

Abstract

Objective: To examine the expression of alpha-crystallin, a small heat-shock protein family, and apoptosis in retinal neoplastic cells.

Methods: Thirteen enucleated globes were included in this study, 1 with retinocytoma and 12 with retinoblastoma. Formalin-fixed paraffin-embedded tissue sections were processed for immunohistochemistry with alpha-crystallin antibodies. Apoptotic cells were detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method.

Results: In the retinocytoma, alphaA-crystallin was expressed in the cytoplasm of all tumor cells, whereas alphaB-crystallin immunoreactivity was only weakly positive. Apoptotic cells were rarely noted in retinocytoma cells; the apoptotic index was 0.29. Examination of the retinoblastoma globes revealed 6 cases (50%) that were strongly positive for alphaA-crystallin. The mean (SD) apoptotic indices in the strongly and weakly positive cases were 3.55 (2.61) and 7.50 (2.61), respectively. The apoptotic index was significantly higher in those cases that were weakly positive for alphaA-crystallin than in those that were strongly positive (P < .05). No correlation was observed between apoptotic index and alphaB-crystallin immunoreactivity, although 50% of retinoblastomas were strongly positive for alphaB-crystallin.

Conclusions: The alphaA- and alphaB-crystallins are expressed in retinoblastomas, and alphaA-crystallin expression may prevent apoptosis of neoplastic cells. Clinical Relevance Suppression of alphaA-crystallin may be useful in controlling tumor growth.

Figure 1

Detection of apoptotic cells in retinoblastoma tissues using confocal microscopy. A, The presence of viable tumor cells can be confirmed using DAPI nuclear staining. B, C, In viable tumor tissues, apoptotic cells are clearly detected by a red signal.

Figure 2

A, Hematoxylin & eosin staining, B, D, DAPI nuclear staining (blue), and C, D, immunodetection of αA-crystallin (green) in the normal retina. A,B, Three nuclear layers consisting of ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) are clearly confirmed. C,D, αA-crystallin immunoreactivity is predominantly detected in the cytoplasm of photoreceptors, which is weakly noted in GCL and INL D. E, Negative control shows no specific immunoreaction in the retina.

Figure 3

A, Hematoxylin & eosin staining, B, immunodetection of αA-crystallin and C, αB-crystallin, and D, TUNEL (red)/DAPI nuclear staining (blue) in human retinocytoma. A, Retinocytoma cells represent a relatively uniform nuclear shape with no apparent nuclear atypia. Mitotic figures are not observed in the tumor tissue. B, αA-crystallin is strongly expressed in the cytoplasm of all tumor cells. C, αB-crystallin immunoreactivity is weakly detected in tumor cells. D, Apoptotic cell is rarely detected in retinocytoma tissue (arrow). Bar indicates 50μm.

Figure 4

A, F, Hematoxylin & eosin staining, B, D, F, E, G, I, J, DAPI nuclear staining (blue) and C,D, immunodetection of αA-crystallin (green) and H,I, αB-crystallin (h, i; green), and E,J, TUNEL-positive tumor cells (red) in human retinoblastoma. A-D, αA-crystallin expression is prominent in the cytoplasm of retinoblastoma cells compared to the normal retina adjacent to the tumor (green) in the representative case. E, TUNEL-positive tumor cells are not detected in the αA-crystallin-positive area. F, Undifferentiated retinoblastoma cells show high proliferation with atypical nuclei. H,I, αB-crystallin is homogeneously expressed in the cytoplasm of retinoblastoma cells (green). J, Apoptotic tumor cell is observed in αB-crystallin-positive area (arrow).

Figure 5

A, B, Hematoxylin & eosin staining, and C, Western blot analysis using retinoblastoma proteins from formalin-fixed, and paraffin-embedded tissues. A, 5-μm-thick section shows retinoblastoma before dissection. B, 30-μm-thick serial section reveals dissection of retinoblastoma tissue. Tumor tissue is excised, where non-cancerous retina is remained. Bars equal 2mm. C, Expression of αA-crystallin is clearly detected in retinoblastoma tissues.

Figure 6

Expression of αA-Crystallin protein in human cultured retinoblastoma cells (Y79) treated with different concentration of hydrogen peroxide (H₂O₂). In untreated Y79, αA-crystallin protein expression is hardly detected. αA-crystallin is expressed in Y79 cells treated with 100 μMol, which reaches maximum at 150 μMol. Expression of αA-crystallin is low in Y79 cells exposed to high concentration of H₂O₂.