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Increased p53 Protein Does Not Correlate to p53 Gene Mutations in Microdissected Human Testicular Germ Cell Tumors
i)O22-5347/95/1542-0617$03.00/0
TtiE JOURNAL
Vol. 154.617-621. Auguat 1996 Printed in U S A
OF UROLDGY
Copyright 0 1995 by
AMERICAN
UROLOCICAL ASSOCUTION, INC
INCREASED p53 PROTEIN DOES NOT CORRELATE TO p53 GENE MUTATIONS IN MICRODISSECTED HUMAN TESTICULAR GERM CELL TUMORS NOAH S. SCHENKMAN, ISABELL A. SESTERHENN, LUCILLE WASHINGTON, YUE A0 TONG, CHRISTOPHER M. WEGHORST, GREGORY S. BUZARD, SHIV SRNASTAVA AND JUDD W. MOUL* From the Urology Service, Department of Surgery, Walter Reed Army Medical Center, Washington, D.C., the Center for Prostate D k a a e Research, Uniformed Services University of the Health Sciences, Bethesda, Maryland, the Armed Forms Institute of P a t M w , Washington, D.C., the Laboratory of Comparative Carcinogenesia, N.C.I., F.C.R.D.C., Freakrick, Maryland and B.C.D.P.. PRIIDyn Corpomtwn, Freakrick, Maryland
ABSTRACT
Purpose: To determine if primary testicular germ cell tumors that overexpress p53 tumor suppressor gene protein have p53 gene mutations. Materials and Methods: We examined 30 primary testicular tissues from 26 patients representing two groups. Group one consisted of eleven cases (6 nonseminomatous germ cell tumors and 5 seminomas) in which tissue samples for DNA analysis were microdissected from p& block regions with elevated immunohistochemical staining for p53 protein. Group two consisted of 19 testis tumor tissues which had been fresh frozen and were chosen to correspond to archival tissue specimens exhibiting elevated levels of p53 protein. The DNA was extracted from these tissues and subjected to exon specific amplification by polymerase chain reaction (PCR) and cold single-strand conformation polymorphism (Cold SSCP) analysis. Results: In these cases with elevated p53 protein, no p53 gene exon 5-8 mutations were detected except 1seminoma with a codon 140 silent mutation (no protein alteration). Conclusions: Testicular tumors appear to exhibit elevated levels of wild-type p53 protein, the significance of which is yet to be elucidated. KEY W o r n : testicular neoplasms; genes, p53; protein p53; polymorphism, single stranded eonformational
Germ cell tumors of the testis represent the most common solid tumor in young men aged 15 to 35 years and have an overall incidence of 2 to 4 per 100,000.1~2Although vast improvementshave occurred in the outcome of these patients due to effective chemotherapy, refinements in therapy are needed in low stage nomeminoma to define occult disease and in advanced nomeminoma to identify treatment-rehctory patients.S.4 The discovery of noninvasive prognostic markers to stage and strati@ patients more accurately is needed to advance these goals. To this end, a si@cant number of studies have focused on defining the molecular biology of testis cancer and identifying molecular markers, including the p53 tumor suppressor gene.5 The p53 gene encodes a 393 amino acid sequence located on the short arm of chromosome 17, and mutations of the p53 gene are frequently found in diverse types of human cancers.6 Ninety percent of the p53 mutations found thus far in human cancers occur in the evolutionarily conserved regiom, including exons 5-8.6 In most tissues, the wild type p53 gene e n d e d
protein is present in small amounts and is degraded rapidly, causingit to be virtually undetectable by immunohistochemical (IHC) staining methods. Abnormal p53 protein is stabilized, resulting in a longer half life and implicating mutation of the p53 gene as the cause of this stability. Thus,p53 gene mutations have been inferred from the increased p53 protein on immunohistochemical staining. Studies of the p53 tumor Suppressor gene and protein in testicular cancer have been contradidry.7-17 While virtuaUy all investigators have found that most testicular tumors exhibit increased p53 protein by i m m u n o ~ t a i n i n g ~ * ~ ~ ~ 1 4 ~ 1 ~ ~ 1 7 several studies have not detected p53 gene mutations that would explain the elevated p53 protein.9*11.15Peng et al. postulated that wild type $3 protein hae a prolonged half life in embryonic tissues such as teaticular tumors although they only esamined testis tumors by DNA analysisP T w o studies have simultaneously examined teaticular tumor tissue DNA for pS3 alteratioxm and levels of p53 protein, and the resulta are conflicting. Heimdal et al. found no p53 gene mutations and no p53 protein in an analysis of 12 testis tumors.11 Conversely, Serth et al. studied tissue sections containing cells that exhibited p53 protein and found a significant number of p53 gene mutations.16 In an effort to clarify the apparent paradox with respect to increased p53 protein and concomitant lack of p53 gene mutations, we carefully mirrodissected identical testicular tumor tissues that exhibited elevated p53 protein to determine if these tumor cell populations contained p53 gene mutations. We used polymerase chain reaction (PCR) of p63 exom 5-8 and a recently described method of cold single strand conformation polymorphism, Cold SSCP, to determine whether p53 mutations are the cause of the elevated p63 protein.
Accepted for publication February 28,1995. uesta for reprink De artment of Surgery, Uniformed SerPiL%kversity of the Heal& Sciences, 4301 Jones Bridge Road, Bethesda, M land 20892-4799. This r e s e a was supported b the followin granta: Department of Clinical Investigation,#2873. %alter Reed Center, Washington, D.C. and The Center for Prostate ase Research ro a m grant #0998-901-1022/G190CM,a ro of the Henry M. jacgon Foundation for the Advancement o f s w Medicine, 1401 Rmkville Pike, Suite 600,Rockville, Maryland 20852-3007. The opinions and assertions contained herein are the private views ofthe authors and are not to be eonstrued as refleeting the views of the US.. Army or the Department of Defense. The m n p t of t b ubhcataon does not necessarily reflect the news or pohcaes of the bepartment of Health and Human SeMces, nor does mention of trade names, commercial products, or organizations imply endonrement by the U.S.government. 617
kmgdd
618
p53 PROTEIN AND p53 GENE MUTATIONS IN GERM CELL TUMORS MATERIALS AND METHODS
A total of 30 primary testicular tissues from 26 patients was studied in 2 groups. Group 1 consisted of 11 archival paraffin-embedded tissues that were the identical blocks used in a previous study of immunohistochemical detection of p53 protein in testicular cancer.17 These 11 cases included 6 nonseminomatous germ cell tumors (NSGCT) and 5 seminomas that exhibited 3' (51to 75% of malignant cells staining) or 4' (76 to 100%) staining for p53 protein. Areas of heavy concentration of p53 positive tumor regions were identified, and corresponding areas of the paraffin embedded tissue blocks were microdissected and placed into plastic Eppendorf tubes as previously described18 (fig. 1).Deoxyribonucleicacid extraction was carried out by a method similar to one described recently.*g Ethanol precipitated DNA was resuspended in 100 pl. of buffer [(lo mM. Tris pH 7.5, 1 mM. EDTA) (TE)]. This product was used directly for PCR amplifications without further concentration or purification. Group 2 consisted of 19 additional fresh-frozen primary testicular tissues from 16 patients which were obtained from the Walter Reed Army Medical Center, Washington, D.C., Urology Tissue Bank. These tissues were obtained a t the time of radical orchiectomy and had been snap-frozen in liquid nitrogen within 1hour f i r orchiectomy. The surgeon had bisected the diseased testis and removed approximately 1cc of tumorous or adjacent normal testicular tissue. These specimens included 10 seminomas, 4 NSGCT, 2 benign tumors (adenomatoid tumor, epidermoid cyst) and 3 areas of adjacent normal testes from these tumors (2 from seminomas, 1 from NSGCT). The high molecular weight DNA was extracted from the fresh-frozen tumor/normal tissues by proteinase K digestion followed by phenollchloroform extraction and ethanol precipitation. These cases had also been analyzed in our previous immunohistochemistry study17 but were not, however, the same tissue as in group 1. One of the NSGCT patients in group 2 was also included in group 1. Polymerase chain reaction /single-stranded conformation polymorphism. The DNA from 19 frozen specimens and 11 paraffinembedded specimens was handled in a similar fashion. p53 exons 5-8 were amplified with primers used in defining the Cold-SSCP procedure.20The 100 pl. PCR reaction contained 2 pl. of template DNA (100 ng.) from the frozen tissue or 4 pl. DNA from the paraffin tissues, 4 pl. (10 to 15 pmol.) of each primer, 10 pl. of 10 x Taq buffer, 0.5 pl.
of 25 mM. dNTP, 0.5 pl. Taq DNA polymerase (Life Technology, 2.5 units) and double-filtered (0.2 pm. disc filter), double-autoclaved deionized distilled water. The PCR reaction mixtures were overlayed with 100 J. of mineral oil, and PCR conditions were set a t 95C, 30 seconds; 60C, 45 seconds; 72C, 1 minute for 40 cycles, then 72C, 5 minutes. The products were stored a t 4C. Positive controls (human placenta DNA) and negative controls (no template) were used for each set of PCR amplifications. All PCR products were checked on 2.5% agarose gels with ethidium bromide staining. The method of Hongyo e t a1.20 was used to perform ColdSSCP on the PCR products of exons 5-8. Five microliters (5 pl.) of PCR product was mixed with 12.6 pI. of 1 X TBE, 2 4. of sample loading buffer containing 15%Ficoll (w/v), 0.25% (wh) bromophenol blue and 0.25% (w/v) xylene cyanol, and 4 pl. of 1 M. methylmercuryhydroxide (Johnson Matthey, Massachusetts). To generate single strand DNA the mixture was incubated at 85C for 5 minutes and chilled in an ice bath. The samples were briefly centrifuged and electrophoresed on 20% polyacrylamide gel (Novex, San Diego, California) at a constant temperature of 1.25 X TBE buffer. Specific temperature and duration of electrophoresis were used for different exons: exon 5: 2 hours 30 minutes at 25C; exon 6: 3 hours at 15C; exon 7: 5 hours a t 1OC; exon 8: 1hour 45 minutes a t 25C for DNA derived from frozen tissue, 3 hours at 1OC for DNA derived from paraffin-embedded tissue. Polymerase chain reaction products of p53 exon 7 with wild type DNA sequence and with heterozygous or hemizygous mutation in codon 245 GGC -+ GAC were run a s controls. Polymerase chain reaction products derived from human placenta DNA for each p53 exon served as wild type controls. ARer electrophoresis the gels were stained with ethidium bromide, read independently by 2 authors (S.S. and N.S.S.) and then stained with silver staining reagents and again evaluated independently by the same authors. Deoxyribonucleic acid sequencing. To generate template for DNA sequencing, a second PCR reaction was performed with an aliquot of the first PCR product. The sense and antisense primers for the second PCR were the same a s for the first PCR except that one of the primers was biotinylated. The custom synthesis of biotinylated primers was performed by Biosynthesis Inc., Lewisville, Texas. The second PCR reaction conditions were similar to those of the first except that the amplification was only 30 cycles. The 5'-biotinylated single stranded DNA was purified with streptavidin-magnetic beads (Dynal, New York), and DNAs were sequenced with complementary sequencing primers and Sequenase version 2.0 (USB). The details of DNA sequencing by means of biotinylated PCR products have been recently described by our laboratory.21 RESULTS
Polymerase chain r e a c t i o n S S C P analysis. A total of 19 specimens from 16 different patients was analyzed from the DNA derived from fresh frozen tissue, and 11 specimens representing 11 patients were analyzed from the DNA retrieved from the microdissected paraffin blocks. All of the specimens were screened for p53 mutations by PCR followed by Cold SSCP. Any abnormalities detected on Cold SSCP were then checked by repeating PCR from genomic DNA and performing a second Cold SSCP. If an altered mobility pattern was detected, the sample was subjected to direct DNA sequencing. No alterations were detected between exons 5 through 8 by SSCP in any of the 19 specimens (10 seminomas, 4 NSGCT, 3 benign tumors, 2 normal testis) derived from fresh-frozen tissue (fig. 2). In contrast to 2 major bands on SSCP gels for RG. 1. p53 immunohistochemicalstain of testis tumor. Malignant germ cells have dark-staining nuclei (arrow).Mar 'ns of microdis- exons 5 , 7 and 8, exon 6 SSCP pattern exhibited the presence of a third band in all of the specimens analyzed (figs.2 and3). section of malignant cells from adjacent normal tutules delineated by broken line. This additional band for exon 6 persisted despitt. gel p d -
p53 PROTEIN AND p53 GENE MUTATIONS IN GERM CELL TUMORS
619
(ACT -+ACC, thr) which turned out to be a silent mutation (fig. 4). Since a study22 of sarcomas revealed that MDM2 gene amplification correlated with the presence of the wild type p53 gene, we have also evaluated the testis tumor D N h from Group 2 for MDM2 gene amplifications. Our studies did not reveal amplification of MDM2 in any of the specimens analyzed (S.S. et al, to be published elsewhere). DISCUSSION
FIG.2. Polymerase chain reactioniCold SSCP analysis of p53 gene exons 5-8 re resented by V-YII from fresh-frozen testis cancer specimens.Afspecimens exhibit s d a r band patterns for each exon indicatin no mutations. P human placenta wild type control. Another wili t (WT) control and mutant (M) in the d o n 245 (GGC +GAC)59 o g o n 7 of p53 gene were amplified with rimem supplied by Clonetech (California)and were used in severafgels to monitor quality of SSCP gels.
The results reported here represent a carehl correlation of p53 exon 5-8 mutational analysis in microdissected areas of testicular tumors exhibiting - nuclear overexpression of the p53 protein. The previous study done by Heimdal et al. correlated IHC findings with DNA analysis in a small cohort (12 patients). The authors found neither p53 gene mutation nor IHC staining in any of this group, although they noted that poor tissue structure may have been responsible for the lack of IHC staining." Most recently, Serth et al. found that 12 of 32 (38%)testicular tumors exhibited p53 protein expression.16 Interestingly, SSCP and DNA sequencing analysis of p53 exon 5-8 confirmed mutations in a subset of these specimens, although details are not evident from the abstract.lB We have found p53 protein expression in at least a portion of the tumor in most testicular cancers,17 a finding similar to those of other investigators.7.S.1214 In exact microdissected
&y::
FIG. 3. Polymerase chain reactiodCold SSCP analysis of om Cl3, represented by V-VIII, from m i c d m e c t d P bedded tesbs cancer specimens. Sample 4, exon 5 demonstrates aberrant band DNA sequencing revealed to be a silent mutation in d o n 140. Sample M represents exon 5 mutation previously char-
actenzed from prostate cancer specimen.
cation of the PCR product, use of 2 different preparations of the PCR primers and use of 3 M e r e n t temperatures for SSCP gel electrophoresis. Thus, in addition to the 2 major collformational forms of single stranded DNAs of exon 6,an additional species is present under OUT experimental conditions. Since this band was present in all of the specimens, including the wild type control, we considered it as a normal pattern for exon 6 under these experimental conditions. Of the 11 microdissected specimens (6NSGCT, 5 seminomas), only 1 seminoma-derived DNA (specimen 4), exhibited aberrant SSCP bands in addition to the wild type specific bands (fig.3). The DNA from this specimen was sequenced directly, and a point mutation was detected in exon 5 at the codon 140
mG.
4. hgyribnueleic +d seqwne ofeample 4 d d d t y p (h) control in region includmg codon 140. h n c e of mutant band in sample 4 ia shown by arrow.
620
p53 PROTEIN AND p53 GENE MUTATIONS IN GERM CELL TUMORS
tissue areas of p53 protein nuclear expression, we now I-eported that the p53 in F9 cells exhibited a normal apoptotic present the evidence of no significant p53 gene mutations in Imesponse to DNA damaging agents. exons 5 through 8. Peng et al. similarly found no p53 gene mutations in exons REFERENCES 2 and 4 through 11 in 22 testicular tumors.9 They speculated from previous work on p53 protein expression in testis tu1. Nikas, P., Champion, A. E. and Fox, M.: Germ cell tumors of testis: prognostic factors and results. Eur. Urol., 18: 242, 1990. mom7 that such tumors overexpressed wild type p53. Peng et 2. Richie, J . P.: Neoplasms of the testis. In: Campbell's Urology. 6th al.9 and 0thers"J-17 had not performed protein and gene edition. Edited by P. C. Walsh, A. B. Retik, T. A. Stamey and analysis on the exact same population of cells within a tumor. E. D. Vaughn, Jr. Philadelphia: W. B. Sanders Co., pp. 1222With the known marked heterogeneity of germ cell tissues 1263, 1992. within a testicular tumor, our analysis was necessary to 3. Einhorn, L. H.: Treatment of testicular cancer: a new and imascertain the nature of p53 activation within individual tuproved model. J . Clin. Oncol., 8: 1777. 1990. mor areas. We have now confirmed that, despite clonal areas 4. Mod, J. W.: Pitfalls in the management of testicular c a n c e ~ of testicular tumors harboring p53 protein nuclear exprespatients and complications of therapy. In: Urology Annual. Edited by S. N. Rous. New York: Norton, pp. 161-197, 1992. sion, no significant p53 gene mutations within exons 5-8 5. Moul, J. W.: Molecular biology of testicular cancer. In: Problem exist. in Urology. Edited by J.W. Moul. Philadelphia: Lippineott, Although p53 protein overexpression in tumor tissues was vol. 8, no. 1, pp. 1-11, 1994. originally believed to represent gene mutation which would 6. Harris,C. C. and Hollstein, M.: Clinical implications of p53 increase the p53 protein half life and permit detection,6 more tumor-suppressor gene. N. Engl. J. Med., 329 1318, 1993. recent evidence suggests that this is not always the case. 7. Bartkova, J., Bartek, J. and Lukas, J.: p53 protein alterations in Studies of human breast cancer?3.24 skin cancer,%Hodgkins human testicular cancer including pre-invasive intratubulax disease,26histiocytomas27and lymphomas27-31 frequently degerm-cell neoplasia. Int. J. Cancer, 4 9 196, 1991. 8. Ruther, V., Nummersiek, C., Muller, H. A. G., Bader, H., Rupp, tect increased p53 protein overexpression without gene muW., Rothe, B., Eisenberger, F. and Jipp, P.: Evidence of wild tation. Furthermore, increased p53 protein may be detected and mutant type p53 in human germ cell tumors by hisb in reactive non-neoplastic tissues.32 The most common explachemical staining. Tumordiagn. Ther., 13: 213, 1992. nation given for this phenomenon is that increased p53 pro9. Peng, H. Q., Hogg, D., Malkin, D., Balley, D., Gallie, B. L., tein expression may be present in certain tissues that exhibit Bulbul, M., Jewett, M., Buchanan, J. and Goss. P. E.: Mutarapid cell proliferation although the exact mechanism of this tions of the p53 gene do not occur in testis cancer. Cancer Res., is yet to be elucidated.* 53: 3574, 1993. That this phenomenon is also seen in human testicular 10. Wei, Y. D., Jaifu, Z., Xi, Q. S., Yongi, M., Xiulong, Z., Daizorg, L. and Jianren, G.: p53 gene mutations in Chinese human tescancer is supported by our data and other experimental reticular seminoma. J. Urol., 150 884, 1993. sults. High levels of wild type p53 are detected in the murine F9 teratocarcinoma cell line,%.= and 8 of 8 human germ cell 11. Heimdal, K, Lother, R., Lyston, S.,Holm, R., Fossl, S. D. and Borresen, A. L.: No germline p53 mutations detected in familtumor lines had increased expression of p53 mRNA.35 p53 is ial and bilateral testicular cancer. Genes Chromosom. Cancer, detectable by immunohistochemistry development due to in6: 92, 1993. creased protein half life.33.36 Peng et al. postulated that in 12. Kuczyk, M. A., Serth, J., Allhoff, E. P., Thon, W. and Jonas, U.: testicular cancer, as in embryonic tissue, p53 protein has an p53 mutant gene expression in archival material of mature increased half Life and may also be transcribed at higher teratoma of the testis. J. Urol., abstract, 1 4 9 310A, 1993. levekS Fleischhacker et al. had speculated that the MDM-2 13. Ulbright, T., Orazi, A., deRiese, W., Messemer, U. and Eble, J.: The relationship of p53 PCNA and S-phase in nonseminomaoncogene may be binding to p53 in testicular tumors as in tous germ cell tumors. Lab Invest., abstract, 68: A71, 1993. sarcomas, but no amplification of MDM-2 was detected.16 We have confirmed this by also finding no amplification of the 14. Schultz, D.S., Linden, M. D. and Konnan, H. J.: Immunohistologic characterization of tumor proliferation and p53 expreaMDM-2 gene in our specimens (unpublished data). Finally a sion in nonseminomatous germ cell tumors. Lab. Invest., abtransgenic mouse model created to measure p53 gene expresstract, 88:69, 1993. sion in vivo found a high level of expression in the testes, and 15. E'leischhacker, M., Strohmeyer, T., Imai, Y., Slamon, D. J. and the authors speculated that p53 protein is necessary in the Koeffler, H. P.: Mutations o f the p53 gene are not detectable in human testicular tumors. Mod. Pathol., 7: 435, 1994. meiotic processes of spermatogenesis.37 It is unclear how and 16. Serth, J., Kuczyk, M. A,, Derendorf, L., Jonasson, J., AlLhoff, if this is related to testicular carcinogenesis. E. P. and Jonas, U.: Identification of frequent p53 gene alterFrom our work and that of othersg.15.17 it now appears ations in testicular cancer by in situ molecular genetic and clear that testicular tumors exhibit elevated wild-type p53, immunohistochemical analysis. J. Urol., abstract, 151: 4 0 8 4 but the s i w c a n c e of this finding is yet to be determined. 1994. p53 protein expression may not indicate an abnormal func- 17. Lewis, D. J., Sesterhenn, I. A., McCarthy, W. F. and Moul, J. W.: tion and may merely represent an epiphenomenon. ConImmunohistochemical expression of p53 tumor suppressor versely, increased p53 protein may confer a selective growth gene protein in adult germ testis tumors: clinical correlation in stage 1 disease. J. Urol., 152 418, 1994. advantage on testicular cancer cell clones by upregulating the expression of cell growth promoting genes, such as cellu- 18. Gaddipati, J . P., McLeod, D. G., Heidenberg, H. B., Sesterhenn, I. A., Finger, M. J., Mod, J . W. and Srivastava, S.: Frequent lar proto-oncogenes. In this regard, expression of the MDM2 detection of codon 877 mutation in the androgen receptor gene gene known to be regulated by p53 needs to be evaluated in advanced prostate cancer. Cancer Res., 54: 2861, 1994. further in testicular tumors. Finally, the apparent increased 19. Moul, J . W.. Lance, R. S., Freidrichs, P. A., Theune, S. M. and levels of wild type p53 protein in testis tumors may represent Chang, E. H.: Infrequent ras oncogene mutations in human inactive forms of the protein stabilized by its interaction with prostate cancer. Prostate, 2 0 327, 1992. a cellular protein. Recent work by Lutzker and Levine38 on 20. Hongyo, T., Buzard, G. S., Calvert, R. J. and Weghorst, C. M.: Cold SSCP: a simple, rapid and non-radioactive method for murine teratocarcinoma cells suggests that, although wild optimized single-strand conformation allomorphism analyses. type p53 is present in these cells, it is defective in its tranNucleic Acids Res., 21: 3637. 1993. scription factor activity. This defect in the transcription factor activity of p53 in F 9 cells can be relieved by protein 21. Heidenberg, H., Sesterhenn, 1. A., Gaddipati, d . , Weghorst, C., Buzard, G. S., Mod, J. W. and Srivastava. S.: Alterations Of synthesis inhibitors, which suggests that a short-lived protumor suppressor gene p53 in a high fraction of treatment tein somehow affects the activity of the wild type p53. It is resistant urostate cancer. ,I. in . Urol... . .. Dress not clear how this interaction may lead to the increased half 22. Oliner, J. D., K~nzler,K. W., Meltzer, P. S..- -George, D. L. and life of the wild tm - - D53. . However. the same studv< also Vogelstein, B.: Amplification of a gene encoding a p53 a s k ~~
~
r - -
p53 PROTEIN AND p53 GENE MUTATIONS IN GERM CELL TUMORS ated protein in human sarcomas. Nature, 358 80, 1992. 23. Coles, C., Condie, A., Chetty, V., Steel, C. M., Evans, H. J., and Prosser, J.: p53 mutations in breast cancer. Cancer Res., 5 2 5291, 1992. 24. Moll, U. M., Riou, G. and Levine, A. J.: Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion. Proc. Natl. Acad. Sci. U.S.A., 89: 7262, 1992. 25. Ziegler, A., Leffell, 0. J., Kunala, S., et al: Mutational hotspots due to sunlight in the p53 gene of nonmelanoma skin cancers. Proc. Natl. Acad. Sci. U.S.A., 90: 4216, 1993. 26. Gupta, R. IL, Patel, K., Bodmer, W. F. and Bodmer, J. G.: Mutation'of p53 in primary biopsy material and cell lines from Hodgkins disease. Proc. Natl. Acad. Sci. U.S.A., 90: 2817, 1993. 27. Soini, Y., Vahakangas, K., Nuorva, K., Kamel, D., Lane, D. P. and Pakko, P.: p53 immunohistochemistry in malignant fibrous histiocytomas and other mesenchymal tumors. J. Pathol., 1Bs: 29, 1992. 28. Gaidano, G., Bellerini, P., Gong, J. Z., Inghirami, G., Neri, A, Newcomb, E. W., Magrath, I. T., Knowles, D. M. and DellaFavera, R.: p53 mutations in human lymphoid malignancies: association with Burkitt lymphoma and chronic lymphocytie leukemia. Roc. Natl. Acad. Sci. U.S.A., 88: 5413, 1991. 29. Nakamura, H., Said, J., Miller, C. W.and Koeffler, H. P.: p53 mutations and immunobistochemistry in AIDS-related lymphomas. Blood, 8 2 920, 1993. 30. Cesarman, E., Inghirami, G., Chadburn, A. and Knowles, D. M.: High levels of p53 protein expression do not wrrelate with p53 gene mutations in CD30 (Ki-1) positive anaplastic large cell 1vmDhoma. Am.J. Pathol.. 14311. 1993. 31. M&&hima, A. Y., CesarmL, E., Chadburn, A. and Kuowles, D. M.: Post-thymic T cell lymphomas frequently overexpress p53 protein but infrequently exhibit p53 gene mutations. Am.
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J. Pathol., 144: 573, 1994. 32. Dei Tos, A. P., Doglioni, C., Laurino, L., Barbareschi, M. and Fletcher C. D.: p53 protein expression in non-neoplastic lesions and benign and malignant neoplasm of soft tissue. Histopathology, 2 2 45, 1993. 33. Chandrasekaran, K, McFarland, W., Simmons, D. T., Dziadek, M., Gurney, E. G. and Mora, P. T.:Quatititation and characterization of a species-specltic and embryo stage-dependent 55-kilodaltonphosphoprotein also present in cells transformed by simian virus 40. Proc. Natl. Acad. Sci. U.S.A., 7 8 6953, 1981. 34. Oren, M., Reich, N. C. and Levine, A. J.: Regulation of the $3 tumor antigen in teratocarcinoma eella and their difFerentiated progeny. Mol. Cell. Biol., 2 443, 1982. 35. T e d , H.,Furbase, R., Casper, J., Lyons, J., Bartram, C. R., Schmoll, H. and Bronaon, D. L.:Cellular oncogenes in human teratmarcinoma cell lines.Int. J. Androl., 1 3 377,1990. 36. Louis, J. M., McFarland, V. W., May, P. and Mora,P. T.: The phosphoprotein p53 is down-regulated p b t r a u s c r i p t i o d y during embryogenesisin vertebrates. Biochem. Biophys. Aeta. SBO: 395, 1988. 37. Almon, E., Goldfinger, N., Kapon, A, Schwartz, D., Levine. A. J. and Ratter, V.: Testicular tissuespecific expression of the p53 suppressor gene. Devel. Biol., 1W. 107, 1993. 38. Lutzker, S. and Levine, A: The regulation of p63 pf trauscn'P tional activity in testicular teratocarcinomaa (abstract). FiRh Meeting on the Molecular Basis of Cancer, Frederick, Maryland, June 1994. 39. Srivastava, S., Zou, Z., Pirollo, K.. Blattner, W. and Chang, E.H.: Germ-line transmission of a mutated p53 gene in a -cancerprone family with Li-Fraumeni syndrome. Nature, s48:747, 1990.
TtiE JOURNAL
Vol. 154.617-621. Auguat 1996 Printed in U S A
OF UROLDGY
Copyright 0 1995 by
AMERICAN
UROLOCICAL ASSOCUTION, INC
INCREASED p53 PROTEIN DOES NOT CORRELATE TO p53 GENE MUTATIONS IN MICRODISSECTED HUMAN TESTICULAR GERM CELL TUMORS NOAH S. SCHENKMAN, ISABELL A. SESTERHENN, LUCILLE WASHINGTON, YUE A0 TONG, CHRISTOPHER M. WEGHORST, GREGORY S. BUZARD, SHIV SRNASTAVA AND JUDD W. MOUL* From the Urology Service, Department of Surgery, Walter Reed Army Medical Center, Washington, D.C., the Center for Prostate D k a a e Research, Uniformed Services University of the Health Sciences, Bethesda, Maryland, the Armed Forms Institute of P a t M w , Washington, D.C., the Laboratory of Comparative Carcinogenesia, N.C.I., F.C.R.D.C., Freakrick, Maryland and B.C.D.P.. PRIIDyn Corpomtwn, Freakrick, Maryland
ABSTRACT
Purpose: To determine if primary testicular germ cell tumors that overexpress p53 tumor suppressor gene protein have p53 gene mutations. Materials and Methods: We examined 30 primary testicular tissues from 26 patients representing two groups. Group one consisted of eleven cases (6 nonseminomatous germ cell tumors and 5 seminomas) in which tissue samples for DNA analysis were microdissected from p& block regions with elevated immunohistochemical staining for p53 protein. Group two consisted of 19 testis tumor tissues which had been fresh frozen and were chosen to correspond to archival tissue specimens exhibiting elevated levels of p53 protein. The DNA was extracted from these tissues and subjected to exon specific amplification by polymerase chain reaction (PCR) and cold single-strand conformation polymorphism (Cold SSCP) analysis. Results: In these cases with elevated p53 protein, no p53 gene exon 5-8 mutations were detected except 1seminoma with a codon 140 silent mutation (no protein alteration). Conclusions: Testicular tumors appear to exhibit elevated levels of wild-type p53 protein, the significance of which is yet to be elucidated. KEY W o r n : testicular neoplasms; genes, p53; protein p53; polymorphism, single stranded eonformational
Germ cell tumors of the testis represent the most common solid tumor in young men aged 15 to 35 years and have an overall incidence of 2 to 4 per 100,000.1~2Although vast improvementshave occurred in the outcome of these patients due to effective chemotherapy, refinements in therapy are needed in low stage nomeminoma to define occult disease and in advanced nomeminoma to identify treatment-rehctory patients.S.4 The discovery of noninvasive prognostic markers to stage and strati@ patients more accurately is needed to advance these goals. To this end, a si@cant number of studies have focused on defining the molecular biology of testis cancer and identifying molecular markers, including the p53 tumor suppressor gene.5 The p53 gene encodes a 393 amino acid sequence located on the short arm of chromosome 17, and mutations of the p53 gene are frequently found in diverse types of human cancers.6 Ninety percent of the p53 mutations found thus far in human cancers occur in the evolutionarily conserved regiom, including exons 5-8.6 In most tissues, the wild type p53 gene e n d e d
protein is present in small amounts and is degraded rapidly, causingit to be virtually undetectable by immunohistochemical (IHC) staining methods. Abnormal p53 protein is stabilized, resulting in a longer half life and implicating mutation of the p53 gene as the cause of this stability. Thus,p53 gene mutations have been inferred from the increased p53 protein on immunohistochemical staining. Studies of the p53 tumor Suppressor gene and protein in testicular cancer have been contradidry.7-17 While virtuaUy all investigators have found that most testicular tumors exhibit increased p53 protein by i m m u n o ~ t a i n i n g ~ * ~ ~ ~ 1 4 ~ 1 ~ ~ 1 7 several studies have not detected p53 gene mutations that would explain the elevated p53 protein.9*11.15Peng et al. postulated that wild type $3 protein hae a prolonged half life in embryonic tissues such as teaticular tumors although they only esamined testis tumors by DNA analysisP T w o studies have simultaneously examined teaticular tumor tissue DNA for pS3 alteratioxm and levels of p53 protein, and the resulta are conflicting. Heimdal et al. found no p53 gene mutations and no p53 protein in an analysis of 12 testis tumors.11 Conversely, Serth et al. studied tissue sections containing cells that exhibited p53 protein and found a significant number of p53 gene mutations.16 In an effort to clarify the apparent paradox with respect to increased p53 protein and concomitant lack of p53 gene mutations, we carefully mirrodissected identical testicular tumor tissues that exhibited elevated p53 protein to determine if these tumor cell populations contained p53 gene mutations. We used polymerase chain reaction (PCR) of p63 exom 5-8 and a recently described method of cold single strand conformation polymorphism, Cold SSCP, to determine whether p53 mutations are the cause of the elevated p63 protein.
Accepted for publication February 28,1995. uesta for reprink De artment of Surgery, Uniformed SerPiL%kversity of the Heal& Sciences, 4301 Jones Bridge Road, Bethesda, M land 20892-4799. This r e s e a was supported b the followin granta: Department of Clinical Investigation,#2873. %alter Reed Center, Washington, D.C. and The Center for Prostate ase Research ro a m grant #0998-901-1022/G190CM,a ro of the Henry M. jacgon Foundation for the Advancement o f s w Medicine, 1401 Rmkville Pike, Suite 600,Rockville, Maryland 20852-3007. The opinions and assertions contained herein are the private views ofthe authors and are not to be eonstrued as refleeting the views of the US.. Army or the Department of Defense. The m n p t of t b ubhcataon does not necessarily reflect the news or pohcaes of the bepartment of Health and Human SeMces, nor does mention of trade names, commercial products, or organizations imply endonrement by the U.S.government. 617
kmgdd
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p53 PROTEIN AND p53 GENE MUTATIONS IN GERM CELL TUMORS MATERIALS AND METHODS
A total of 30 primary testicular tissues from 26 patients was studied in 2 groups. Group 1 consisted of 11 archival paraffin-embedded tissues that were the identical blocks used in a previous study of immunohistochemical detection of p53 protein in testicular cancer.17 These 11 cases included 6 nonseminomatous germ cell tumors (NSGCT) and 5 seminomas that exhibited 3' (51to 75% of malignant cells staining) or 4' (76 to 100%) staining for p53 protein. Areas of heavy concentration of p53 positive tumor regions were identified, and corresponding areas of the paraffin embedded tissue blocks were microdissected and placed into plastic Eppendorf tubes as previously described18 (fig. 1).Deoxyribonucleicacid extraction was carried out by a method similar to one described recently.*g Ethanol precipitated DNA was resuspended in 100 pl. of buffer [(lo mM. Tris pH 7.5, 1 mM. EDTA) (TE)]. This product was used directly for PCR amplifications without further concentration or purification. Group 2 consisted of 19 additional fresh-frozen primary testicular tissues from 16 patients which were obtained from the Walter Reed Army Medical Center, Washington, D.C., Urology Tissue Bank. These tissues were obtained a t the time of radical orchiectomy and had been snap-frozen in liquid nitrogen within 1hour f i r orchiectomy. The surgeon had bisected the diseased testis and removed approximately 1cc of tumorous or adjacent normal testicular tissue. These specimens included 10 seminomas, 4 NSGCT, 2 benign tumors (adenomatoid tumor, epidermoid cyst) and 3 areas of adjacent normal testes from these tumors (2 from seminomas, 1 from NSGCT). The high molecular weight DNA was extracted from the fresh-frozen tumor/normal tissues by proteinase K digestion followed by phenollchloroform extraction and ethanol precipitation. These cases had also been analyzed in our previous immunohistochemistry study17 but were not, however, the same tissue as in group 1. One of the NSGCT patients in group 2 was also included in group 1. Polymerase chain reaction /single-stranded conformation polymorphism. The DNA from 19 frozen specimens and 11 paraffinembedded specimens was handled in a similar fashion. p53 exons 5-8 were amplified with primers used in defining the Cold-SSCP procedure.20The 100 pl. PCR reaction contained 2 pl. of template DNA (100 ng.) from the frozen tissue or 4 pl. DNA from the paraffin tissues, 4 pl. (10 to 15 pmol.) of each primer, 10 pl. of 10 x Taq buffer, 0.5 pl.
of 25 mM. dNTP, 0.5 pl. Taq DNA polymerase (Life Technology, 2.5 units) and double-filtered (0.2 pm. disc filter), double-autoclaved deionized distilled water. The PCR reaction mixtures were overlayed with 100 J. of mineral oil, and PCR conditions were set a t 95C, 30 seconds; 60C, 45 seconds; 72C, 1 minute for 40 cycles, then 72C, 5 minutes. The products were stored a t 4C. Positive controls (human placenta DNA) and negative controls (no template) were used for each set of PCR amplifications. All PCR products were checked on 2.5% agarose gels with ethidium bromide staining. The method of Hongyo e t a1.20 was used to perform ColdSSCP on the PCR products of exons 5-8. Five microliters (5 pl.) of PCR product was mixed with 12.6 pI. of 1 X TBE, 2 4. of sample loading buffer containing 15%Ficoll (w/v), 0.25% (wh) bromophenol blue and 0.25% (w/v) xylene cyanol, and 4 pl. of 1 M. methylmercuryhydroxide (Johnson Matthey, Massachusetts). To generate single strand DNA the mixture was incubated at 85C for 5 minutes and chilled in an ice bath. The samples were briefly centrifuged and electrophoresed on 20% polyacrylamide gel (Novex, San Diego, California) at a constant temperature of 1.25 X TBE buffer. Specific temperature and duration of electrophoresis were used for different exons: exon 5: 2 hours 30 minutes at 25C; exon 6: 3 hours at 15C; exon 7: 5 hours a t 1OC; exon 8: 1hour 45 minutes a t 25C for DNA derived from frozen tissue, 3 hours at 1OC for DNA derived from paraffin-embedded tissue. Polymerase chain reaction products of p53 exon 7 with wild type DNA sequence and with heterozygous or hemizygous mutation in codon 245 GGC -+ GAC were run a s controls. Polymerase chain reaction products derived from human placenta DNA for each p53 exon served as wild type controls. ARer electrophoresis the gels were stained with ethidium bromide, read independently by 2 authors (S.S. and N.S.S.) and then stained with silver staining reagents and again evaluated independently by the same authors. Deoxyribonucleic acid sequencing. To generate template for DNA sequencing, a second PCR reaction was performed with an aliquot of the first PCR product. The sense and antisense primers for the second PCR were the same a s for the first PCR except that one of the primers was biotinylated. The custom synthesis of biotinylated primers was performed by Biosynthesis Inc., Lewisville, Texas. The second PCR reaction conditions were similar to those of the first except that the amplification was only 30 cycles. The 5'-biotinylated single stranded DNA was purified with streptavidin-magnetic beads (Dynal, New York), and DNAs were sequenced with complementary sequencing primers and Sequenase version 2.0 (USB). The details of DNA sequencing by means of biotinylated PCR products have been recently described by our laboratory.21 RESULTS
Polymerase chain r e a c t i o n S S C P analysis. A total of 19 specimens from 16 different patients was analyzed from the DNA derived from fresh frozen tissue, and 11 specimens representing 11 patients were analyzed from the DNA retrieved from the microdissected paraffin blocks. All of the specimens were screened for p53 mutations by PCR followed by Cold SSCP. Any abnormalities detected on Cold SSCP were then checked by repeating PCR from genomic DNA and performing a second Cold SSCP. If an altered mobility pattern was detected, the sample was subjected to direct DNA sequencing. No alterations were detected between exons 5 through 8 by SSCP in any of the 19 specimens (10 seminomas, 4 NSGCT, 3 benign tumors, 2 normal testis) derived from fresh-frozen tissue (fig. 2). In contrast to 2 major bands on SSCP gels for RG. 1. p53 immunohistochemicalstain of testis tumor. Malignant germ cells have dark-staining nuclei (arrow).Mar 'ns of microdis- exons 5 , 7 and 8, exon 6 SSCP pattern exhibited the presence of a third band in all of the specimens analyzed (figs.2 and3). section of malignant cells from adjacent normal tutules delineated by broken line. This additional band for exon 6 persisted despitt. gel p d -
p53 PROTEIN AND p53 GENE MUTATIONS IN GERM CELL TUMORS
619
(ACT -+ACC, thr) which turned out to be a silent mutation (fig. 4). Since a study22 of sarcomas revealed that MDM2 gene amplification correlated with the presence of the wild type p53 gene, we have also evaluated the testis tumor D N h from Group 2 for MDM2 gene amplifications. Our studies did not reveal amplification of MDM2 in any of the specimens analyzed (S.S. et al, to be published elsewhere). DISCUSSION
FIG.2. Polymerase chain reactioniCold SSCP analysis of p53 gene exons 5-8 re resented by V-YII from fresh-frozen testis cancer specimens.Afspecimens exhibit s d a r band patterns for each exon indicatin no mutations. P human placenta wild type control. Another wili t (WT) control and mutant (M) in the d o n 245 (GGC +GAC)59 o g o n 7 of p53 gene were amplified with rimem supplied by Clonetech (California)and were used in severafgels to monitor quality of SSCP gels.
The results reported here represent a carehl correlation of p53 exon 5-8 mutational analysis in microdissected areas of testicular tumors exhibiting - nuclear overexpression of the p53 protein. The previous study done by Heimdal et al. correlated IHC findings with DNA analysis in a small cohort (12 patients). The authors found neither p53 gene mutation nor IHC staining in any of this group, although they noted that poor tissue structure may have been responsible for the lack of IHC staining." Most recently, Serth et al. found that 12 of 32 (38%)testicular tumors exhibited p53 protein expression.16 Interestingly, SSCP and DNA sequencing analysis of p53 exon 5-8 confirmed mutations in a subset of these specimens, although details are not evident from the abstract.lB We have found p53 protein expression in at least a portion of the tumor in most testicular cancers,17 a finding similar to those of other investigators.7.S.1214 In exact microdissected
&y::
FIG. 3. Polymerase chain reactiodCold SSCP analysis of om Cl3, represented by V-VIII, from m i c d m e c t d P bedded tesbs cancer specimens. Sample 4, exon 5 demonstrates aberrant band DNA sequencing revealed to be a silent mutation in d o n 140. Sample M represents exon 5 mutation previously char-
actenzed from prostate cancer specimen.
cation of the PCR product, use of 2 different preparations of the PCR primers and use of 3 M e r e n t temperatures for SSCP gel electrophoresis. Thus, in addition to the 2 major collformational forms of single stranded DNAs of exon 6,an additional species is present under OUT experimental conditions. Since this band was present in all of the specimens, including the wild type control, we considered it as a normal pattern for exon 6 under these experimental conditions. Of the 11 microdissected specimens (6NSGCT, 5 seminomas), only 1 seminoma-derived DNA (specimen 4), exhibited aberrant SSCP bands in addition to the wild type specific bands (fig.3). The DNA from this specimen was sequenced directly, and a point mutation was detected in exon 5 at the codon 140
mG.
4. hgyribnueleic +d seqwne ofeample 4 d d d t y p (h) control in region includmg codon 140. h n c e of mutant band in sample 4 ia shown by arrow.
620
p53 PROTEIN AND p53 GENE MUTATIONS IN GERM CELL TUMORS
tissue areas of p53 protein nuclear expression, we now I-eported that the p53 in F9 cells exhibited a normal apoptotic present the evidence of no significant p53 gene mutations in Imesponse to DNA damaging agents. exons 5 through 8. Peng et al. similarly found no p53 gene mutations in exons REFERENCES 2 and 4 through 11 in 22 testicular tumors.9 They speculated from previous work on p53 protein expression in testis tu1. Nikas, P., Champion, A. E. and Fox, M.: Germ cell tumors of testis: prognostic factors and results. Eur. Urol., 18: 242, 1990. mom7 that such tumors overexpressed wild type p53. Peng et 2. Richie, J . P.: Neoplasms of the testis. In: Campbell's Urology. 6th al.9 and 0thers"J-17 had not performed protein and gene edition. Edited by P. C. Walsh, A. B. Retik, T. A. Stamey and analysis on the exact same population of cells within a tumor. E. D. Vaughn, Jr. Philadelphia: W. B. Sanders Co., pp. 1222With the known marked heterogeneity of germ cell tissues 1263, 1992. within a testicular tumor, our analysis was necessary to 3. Einhorn, L. H.: Treatment of testicular cancer: a new and imascertain the nature of p53 activation within individual tuproved model. J . Clin. Oncol., 8: 1777. 1990. mor areas. We have now confirmed that, despite clonal areas 4. Mod, J. W.: Pitfalls in the management of testicular c a n c e ~ of testicular tumors harboring p53 protein nuclear exprespatients and complications of therapy. In: Urology Annual. Edited by S. N. Rous. New York: Norton, pp. 161-197, 1992. sion, no significant p53 gene mutations within exons 5-8 5. Moul, J. W.: Molecular biology of testicular cancer. In: Problem exist. in Urology. Edited by J.W. Moul. Philadelphia: Lippineott, Although p53 protein overexpression in tumor tissues was vol. 8, no. 1, pp. 1-11, 1994. originally believed to represent gene mutation which would 6. Harris,C. C. and Hollstein, M.: Clinical implications of p53 increase the p53 protein half life and permit detection,6 more tumor-suppressor gene. N. Engl. J. Med., 329 1318, 1993. recent evidence suggests that this is not always the case. 7. Bartkova, J., Bartek, J. and Lukas, J.: p53 protein alterations in Studies of human breast cancer?3.24 skin cancer,%Hodgkins human testicular cancer including pre-invasive intratubulax disease,26histiocytomas27and lymphomas27-31 frequently degerm-cell neoplasia. Int. J. Cancer, 4 9 196, 1991. 8. Ruther, V., Nummersiek, C., Muller, H. A. G., Bader, H., Rupp, tect increased p53 protein overexpression without gene muW., Rothe, B., Eisenberger, F. and Jipp, P.: Evidence of wild tation. Furthermore, increased p53 protein may be detected and mutant type p53 in human germ cell tumors by hisb in reactive non-neoplastic tissues.32 The most common explachemical staining. Tumordiagn. Ther., 13: 213, 1992. nation given for this phenomenon is that increased p53 pro9. Peng, H. Q., Hogg, D., Malkin, D., Balley, D., Gallie, B. L., tein expression may be present in certain tissues that exhibit Bulbul, M., Jewett, M., Buchanan, J. and Goss. P. E.: Mutarapid cell proliferation although the exact mechanism of this tions of the p53 gene do not occur in testis cancer. Cancer Res., is yet to be elucidated.* 53: 3574, 1993. That this phenomenon is also seen in human testicular 10. Wei, Y. D., Jaifu, Z., Xi, Q. S., Yongi, M., Xiulong, Z., Daizorg, L. and Jianren, G.: p53 gene mutations in Chinese human tescancer is supported by our data and other experimental reticular seminoma. J. Urol., 150 884, 1993. sults. High levels of wild type p53 are detected in the murine F9 teratocarcinoma cell line,%.= and 8 of 8 human germ cell 11. Heimdal, K, Lother, R., Lyston, S.,Holm, R., Fossl, S. D. and Borresen, A. L.: No germline p53 mutations detected in familtumor lines had increased expression of p53 mRNA.35 p53 is ial and bilateral testicular cancer. Genes Chromosom. Cancer, detectable by immunohistochemistry development due to in6: 92, 1993. creased protein half life.33.36 Peng et al. postulated that in 12. Kuczyk, M. A., Serth, J., Allhoff, E. P., Thon, W. and Jonas, U.: testicular cancer, as in embryonic tissue, p53 protein has an p53 mutant gene expression in archival material of mature increased half Life and may also be transcribed at higher teratoma of the testis. J. Urol., abstract, 1 4 9 310A, 1993. levekS Fleischhacker et al. had speculated that the MDM-2 13. Ulbright, T., Orazi, A., deRiese, W., Messemer, U. and Eble, J.: The relationship of p53 PCNA and S-phase in nonseminomaoncogene may be binding to p53 in testicular tumors as in tous germ cell tumors. Lab Invest., abstract, 68: A71, 1993. sarcomas, but no amplification of MDM-2 was detected.16 We have confirmed this by also finding no amplification of the 14. Schultz, D.S., Linden, M. D. and Konnan, H. J.: Immunohistologic characterization of tumor proliferation and p53 expreaMDM-2 gene in our specimens (unpublished data). Finally a sion in nonseminomatous germ cell tumors. Lab. Invest., abtransgenic mouse model created to measure p53 gene expresstract, 88:69, 1993. sion in vivo found a high level of expression in the testes, and 15. E'leischhacker, M., Strohmeyer, T., Imai, Y., Slamon, D. J. and the authors speculated that p53 protein is necessary in the Koeffler, H. P.: Mutations o f the p53 gene are not detectable in human testicular tumors. Mod. Pathol., 7: 435, 1994. meiotic processes of spermatogenesis.37 It is unclear how and 16. Serth, J., Kuczyk, M. A,, Derendorf, L., Jonasson, J., AlLhoff, if this is related to testicular carcinogenesis. E. P. and Jonas, U.: Identification of frequent p53 gene alterFrom our work and that of othersg.15.17 it now appears ations in testicular cancer by in situ molecular genetic and clear that testicular tumors exhibit elevated wild-type p53, immunohistochemical analysis. J. Urol., abstract, 151: 4 0 8 4 but the s i w c a n c e of this finding is yet to be determined. 1994. p53 protein expression may not indicate an abnormal func- 17. Lewis, D. J., Sesterhenn, I. A., McCarthy, W. F. and Moul, J. W.: tion and may merely represent an epiphenomenon. ConImmunohistochemical expression of p53 tumor suppressor versely, increased p53 protein may confer a selective growth gene protein in adult germ testis tumors: clinical correlation in stage 1 disease. J. Urol., 152 418, 1994. advantage on testicular cancer cell clones by upregulating the expression of cell growth promoting genes, such as cellu- 18. Gaddipati, J . P., McLeod, D. G., Heidenberg, H. B., Sesterhenn, I. A., Finger, M. J., Mod, J . 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M.: Cold SSCP: a simple, rapid and non-radioactive method for murine teratocarcinoma cells suggests that, although wild optimized single-strand conformation allomorphism analyses. type p53 is present in these cells, it is defective in its tranNucleic Acids Res., 21: 3637. 1993. scription factor activity. This defect in the transcription factor activity of p53 in F 9 cells can be relieved by protein 21. Heidenberg, H., Sesterhenn, 1. A., Gaddipati, d . , Weghorst, C., Buzard, G. S., Mod, J. W. and Srivastava. S.: Alterations Of synthesis inhibitors, which suggests that a short-lived protumor suppressor gene p53 in a high fraction of treatment tein somehow affects the activity of the wild type p53. It is resistant urostate cancer. ,I. in . Urol... . .. Dress not clear how this interaction may lead to the increased half 22. Oliner, J. D., K~nzler,K. W., Meltzer, P. S..- -George, D. L. and life of the wild tm - - D53. . However. the same studv< also Vogelstein, B.: Amplification of a gene encoding a p53 a s k ~~
~
r - -
p53 PROTEIN AND p53 GENE MUTATIONS IN GERM CELL TUMORS ated protein in human sarcomas. Nature, 358 80, 1992. 23. Coles, C., Condie, A., Chetty, V., Steel, C. M., Evans, H. J., and Prosser, J.: p53 mutations in breast cancer. Cancer Res., 5 2 5291, 1992. 24. Moll, U. M., Riou, G. and Levine, A. J.: Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion. Proc. Natl. Acad. Sci. U.S.A., 89: 7262, 1992. 25. Ziegler, A., Leffell, 0. J., Kunala, S., et al: Mutational hotspots due to sunlight in the p53 gene of nonmelanoma skin cancers. Proc. Natl. Acad. Sci. U.S.A., 90: 4216, 1993. 26. Gupta, R. IL, Patel, K., Bodmer, W. F. and Bodmer, J. G.: Mutation'of p53 in primary biopsy material and cell lines from Hodgkins disease. Proc. Natl. Acad. Sci. U.S.A., 90: 2817, 1993. 27. Soini, Y., Vahakangas, K., Nuorva, K., Kamel, D., Lane, D. P. and Pakko, P.: p53 immunohistochemistry in malignant fibrous histiocytomas and other mesenchymal tumors. J. Pathol., 1Bs: 29, 1992. 28. Gaidano, G., Bellerini, P., Gong, J. Z., Inghirami, G., Neri, A, Newcomb, E. W., Magrath, I. T., Knowles, D. M. and DellaFavera, R.: p53 mutations in human lymphoid malignancies: association with Burkitt lymphoma and chronic lymphocytie leukemia. Roc. Natl. Acad. Sci. U.S.A., 88: 5413, 1991. 29. Nakamura, H., Said, J., Miller, C. W.and Koeffler, H. P.: p53 mutations and immunobistochemistry in AIDS-related lymphomas. Blood, 8 2 920, 1993. 30. Cesarman, E., Inghirami, G., Chadburn, A. and Knowles, D. M.: High levels of p53 protein expression do not wrrelate with p53 gene mutations in CD30 (Ki-1) positive anaplastic large cell 1vmDhoma. Am.J. Pathol.. 14311. 1993. 31. M&&hima, A. Y., CesarmL, E., Chadburn, A. and Kuowles, D. M.: Post-thymic T cell lymphomas frequently overexpress p53 protein but infrequently exhibit p53 gene mutations. Am.
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