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Nuclear exclusion of wild-type p53 in immortalized human retinoblastoma cells
C L Schlamp1, G L Poulsen, T M Nork
1Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison 53792, USA.
Background:
Retinoblastoma is the most common childhood tumor of the eye, arising from cells that are defective in both copies of the retinoblastoma susceptibility gene (RB1). Most retinoblastoma tumor cells eventually undergo programmed cell death (i.e., apoptosis); however, some cells can acquire the ability to metastasize and become immortal. Transfection of immortal retinoblastoma cells with DNA sequences encoding wild-type p53 protein induces cell death, suggesting that the loss of both RB1 and p53 functions may be required for cell immortalization. We have examined this possibility by characterizing the p53 protein and messenger RNA in six independently isolated, immortalized retinoblastoma cell lines.
Methods:
Western blotting methods were used to assess p53 protein level in each cell line, and Cleavase Fragment-Length Polymorphism analysis of complementary DNAs was used to screen for mutations in p53 messenger RNA. Localization of p53 protein in cells of the immortalized lines and in specimens of retinoblastoma tumors was achieved by means of indirect immunofluorescence and immunocytochemistry, respectively.
Results:
All six immortalized cell lines expressed wild-type p53 messenger RNA and high levels of p53 protein. Although p53 is normally a nuclear protein, the p53 in four of the six cell lines was located predominately in the cytoplasm; in the remaining two cell lines, p53 was localized in both the nucleus and the cytoplasm. Cytoplasmic localization of p53 in retinoblastoma tumor specimens was rare and usually restricted to cells that had invaded adjacent ocular tissues, indicative of the early stages of metastasis.
Conclusions:
Some immortalized retinoblastoma cells may exhibit p53 dysfunction through nuclear exclusion of wild-type p53 protein.
Insights
Immortalized retinoblastoma cells can show p53 (tumor suppressor) dysfunction. This occurs when the wild-type p53 protein is excluded from the nucleus, hindering its function in preventing cancer progression.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Retinoblastoma, a common childhood eye tumor, arises from RB1 gene defects.
- Tumor cells typically undergo apoptosis, but some metastasize and become immortal.
- Loss of both RB1 and p53 functions may be crucial for retinoblastoma cell immortalization.
Purpose of the Study:
- To investigate the role of p53 protein and messenger RNA in immortalized retinoblastoma cell lines.
- To determine if p53 dysfunction contributes to retinoblastoma cell immortalization.
Main Methods:
- Western blotting to assess p53 protein levels.
- Cleavase Fragment-Length Polymorphism analysis to detect p53 messenger RNA mutations.
- Indirect immunofluorescence and immunocytochemistry to determine p53 protein localization.
Main Results:
- All six immortalized retinoblastoma cell lines expressed wild-type p53 messenger RNA and high p53 protein levels.
- In four of six cell lines, p53 protein was predominantly located in the cytoplasm.
- In the remaining two cell lines, p53 was found in both the nucleus and cytoplasm.
- Cytoplasmic p53 localization was rare in retinoblastoma tumors, mainly seen in cells invading adjacent tissues.
Conclusions:
- Immortalized retinoblastoma cells can display p53 dysfunction.
- This dysfunction may result from the nuclear exclusion of wild-type p53 protein.
- Nuclear exclusion of p53 could be a mechanism contributing to retinoblastoma cell immortalization and metastasis.