ELSEVIER l Correspondence ~53 PROTEIN EXPRESSION OR PROTEIN FUNCTION? RE: AWWAD ET AL,. IJROBP 34(2):323-332; 1996 To the Editor: Experimental studies in vitro have documented that the normal cellular response to ionizing radiation includes increased expression of the wild-type p53 (WTp53) tumor suppressor protein that may determine radiation-induced cell cycle arrest, and in some cell types. apoptosis (7). Furthermore, these “normal” cellular functions are often absent in cells expressing a mutant p53 (MTp53) protein. A number of in virro studies, including our own, have observed relative radioresistance in human or rodent tumor cells that lack WTp53 protein, or express MTp53 proteins, presumed due to a dominant-negative effect of certain MTp53 proteins over the normal function of WTp53 proteins in heterozygote cell lines (6, 12, 14). Among these studies, correlations do exist between the level of MTp53 protein expression and radioresis- tance as determined by clonogenic assays in vitro, and may be further modified by the coexpression of cooperating oncogenes, such as the H- ras gene (6). Acquired radioresistance has also been observed experi- mentally in human fibroblasts transfected with HPV-E6/E7 oncopro- teins ( 16). These studies support the hypothesis that an abrogated Gl cell cycle checkpoint may be required for acquired radioresistance in tumor cells. Previous radiobiologic studies of head and neck cancer cell lines have not addressed the above issues, although one study did ob- serve a trend towards acquired radioresistance among cell lines which expressed a MTp5 3 sequence ( 3 ) We read, with interest, the recent report by Awwad et al. ( 1 ) who observed that ~53 protein overexpression in pretreatment tumor speci- mens was not predictive for disease-free survival following radical ra- diotherapy. In this study, ~53 protein expression as measured by immu- nohistochemistry (MC) did not predict for tumor site, tumor stage, or tumor recurrence following radiotherapy. In discussing their results, the authors acknowledge that ~53 protein expression can be altered by mechanisms other than p53 gene mutation in tumor cells (i.e., coex- pressed HPV or mdm-2 sequences), which could potentially confound the results of their IHC-analyses (4, 18). Furthermore, Schin et al. ( 15) have documented the presence of p53-overexpression also among premalignant cells in selected head and neck mmors. It will be of interest to see whether a strong concordance exists between the IHC analysis and future DNA sequencing studies by Awwad and co-workers ( 1 ), as recent data supports the use of a rigorous DNA sequencing endpoint in studies of clinicopathologic outcome (2, 5, 9). The use of assays other than DNA sequencing may explain the disparate conclusions reached by a number of groups regarding the potential role of the p53 gene as a predictor of radiotherapeutic response among human brain, head and neck, breast, and lung cancers (2, 9, 10. 11, 17). It is important to note that not all MTp53 proteins derived from human tumor cells actually acquire the ability to alter WTp53 protein- directed cellular activities-in heterozygote tumor cells which &express MTp53 and WTp53 proteins ( 13 ) However. most human MTn53-ex- pressing tumor cells*subsequdntly lose the expression of the ‘WTp53 allele during tumor progression, which can result in a “loss of heterozy- gosity,” and the sole expression of a MTp53 allele (7). These allelic changes may have important implications for MTp53 protein activity, and cannot be determined by IHC-analyses alone. In summary, although IHC-analyses will provide information regarding the presence or ab- sence of p53 protein “expression.” they will not directly determine the presence or absence of WTp53 protein “function.” A number of potential predictive assaysof radiocurability (i.e., SFzF, , T,, , apoptotic index, and tumor oxygenation) are currently undergoing prospective testing, and studies such as the one by Awwad et al. ( 1) are commendable for their attempts to define potential genetic determi- nants of radiocurability. And yet, ultimately, assaysof proteinfuncrion may be required to determine whether cycle control proteins, such as the p53 or retinoblastoma gene products, can modify tumor cell radiore- sponse in vifro and in viva. Functional assays will undoubtedly involve multi-parameter flow cytometry and irradiated tumor cells in order to simultaneously document changes in cell cycle progression and relative protein expression ( 14). A more thorough understanding of the molecu- lar events which follow WTp53 protein induction following DNA dam- age and their derrangement in MTp53containing cells will no doubt provide further avenues of radiobiologic research, and hopefully allow a direct assessment as to whether these events are of sufficient impor- tance to determine human tumour radiocurability 1Y) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. ROBERT G. Bmsrow, M.D.. M.Sc. SAM BENCHIMOI., Ph.D. RICHARD HILL, Ph.D. Departments of Radiation Oncology and Medical Biophysics University of Toronto, and The Ontario I’ancer Institute/ Princess Margaret Hospital 6 10 University Avenue Toronto, Ontario CANADA MSG 2M9 Awwad, S.; Jaros. E.; Somes, J.: Lunec, J. ~5.3 overexpression in head and neck carcinoma and radiotherapy results. lnt. J. Rad. Oncol. Biol. Phys. 34(2):323-332; 1996. Bergh, J.; Norberg, T.; Sjogren, S.; Lindgren. A.; Holmberg, L. 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