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RT Carter, R Dubielzig, CM H Colitz; Immunohistochemical Staining for TRT, PCNA and p16 In Post-Traumatic Ocular Sarcomas of Cats . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2583.
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Purpose: To evaluate post-traumatic ocular sarcomas of cats immunohistochemically for the presence of TRT (the catalytic subunit of telomerase), PCNA (a marker of cellular proliferation) and p16 (an inhibitor of cellular proliferation). Methods: Twenty-four paraffin-embedded tissue samples from cats documented with ocular sarcoma were sectioned onto Probe-On Plus slides (Fisher). Sections were deparaffinized by a series of xylene rinses and rehydrated through a series of alcohols and 1X PBS rinses. Sections to be stained for TRT were incubated in antigen retrieval solution (DAKO) at 95oC for 40 minutes, then at room temperature for 20 minutes. Antigen retrieval was not utilized for sections stained for PCNA or p16. Immunohistochemical staining was performed using previously described avidin-biotin-peroxidase complex techniques with diaminobenzidine as the chromogen and counterstaining with hematoxylin. Commercially available antibodies to TRT (Calbiochem), PCNA (Stressgen) and p16 (Chemicon) were used at dilutions of 1:500 for TRT and p16, 1:250 for PCNA. Feline tonsil was used as the positive control for TRT, p16 and PCNA. Sections incubated without the primary antibody were used as the negative control. Nuclear staining was used to identify cells positive for TRT and PCNA; cytoplasmic and perinuclear staining were used to identify cells positive for p16. Results: Immunostaining was positive in 15/24 samples (62.5%) for TRT and 22/24 (91.7%) for PCNA. Immunostaining was negative for p16 in 17/24 samples (70.8%); one sample was variably positive for p16. Of the samples that were TRT positive, 14/15 (93.3%) were concurrently PCNA positive and 10/15 (66.7%) were p16 negative. Conclusion: We have found that 66.7% of feline ocular sarcomas with TRT expression also expressed a loss of p16. These results parallel the findings in human literature that cellular immortalization requires increased telomerase activity (indirectly measured through TRT) and loss of cell cycle regulation (p16). The high incidence of PCNA expression reflects the degree of cellular proliferation. We would have expected that more of the samples would have expressed TRT, however, these results may have been affected by sample processing, handing and sectioning.
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