ARTICLE |
Correspondence to: M. Friedrich, Universitäts-Frauenklinik, Gebäude 9, D-66421 Homburg/Saar, Germany..
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We analyzed human Mut-S-Homologon-2 expression in normal endometrial tissue (n = 15) and malignancies of the uterine corpus (n = 40). Human Mut-S-Homologon-2 protein was investigated immunohistochemically on frozen sections, using a highly sensitive streptavidinperoxidase technique and a specific mouse monoclonal antibody (clone FE11). Human Mut-S-Homologon-2 labeling pattern was compared with the staining pattern of the proliferation marker Ki-67 in the same tumors. A human Mut-S-Homologon-2 immunoreactivity score (human Mut-S-Homologon-2-IRS: negative 01; weak 23; moderate 46; strong 812) for semiquantitative analysis of human Mut-S-Homologon-2 expression is presented. In normal endometrial tissue samples we found weak nuclear immunoreactivity for human Mut-S-Homologon-2 in 67%, whereas the remaining 33% were negative for human Mut-S-Homologon-2 (mean human Mut-S-Homologon-2-IRS 1.25 ± 1.29). All malignancies of the uterine corpus analyzed revealed moderate to strong nuclear immunoreactivity (mean human Mut-S-Homologon-2-IRS 9.00, ± 3.16). Human Mut-S-Homologon-2 staining was heterogeneous, with visual differences among individual tumor cells. Expression of human Mut-S-Homologon-2 protein was consistently and strongly upregulated in tumor cells of malignancies of the uterine corpus compared with normal endometrial tissue (human Mut-S-Homologon-2-PP p<0.001; human Mut-S-Homologon-2-IS p<0.001; human Mut-S-Homologon-2-IRS p<0.001). No statistically significant correlation in comparing the labeling patterns for human Mut-S-Homologon-2 with the labeling patterns for Ki-67 (mean percentage of Ki-67-positive tumor cells 22.00% ± 17.20) was observed in malignancies of the uterine corpus (human Mut-S-Homologon-2-PP p=0.443; human Mut-S-Homologon-2-IS p=0.234; human Mut-S-Homologon-2-IRS p=0.173). Our findings indicate that human Mut-S-Homologon-2 is expressed in normal human endometrial tissue and that expression of human Mut-S-Homologon-2 may be of importance for the genetic stability of malignancies of the uterine corpus in vivo. (J Histochem Cytochem 47:113118, 1999).
Key Words: human Mut-S-Homologon-2, DNA repair, malignancies of the uterine, corpus, carcinogenesis, immunohistochemistry
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It was recently demonstrated that microsatellite instability secondary to replication errors can be detected in various malignant human tumors (
Endometrial carcinoma is the most common nonrectal carcinoma in women affected by HNPCC, and microsatellite instability has been observed both in the inherited form and in approximately 20% of presumably sporadic endometrial carcinomas (
The aim of this study was to investigate the expression of human Mut-S-Homologon-2 in normal endometrial tissue and in malignancies of the uterine corpus. We asked the following questions: (a) Do we find human Mut-S-Homologon-2 immunoreactivity in normal endometrial tissue, pointing to a functional importance of this DNA repair enzyme for the genetic stability of endometrial cells in vivo? (b) Do we find changes in the human Mut-S-Homologon-2 staining pattern in malignancies of the uterine corpus, pointing to a contribution of this DNA repair enzyme to the carcinogenesis or genetic stability of them in vivo? (c) Do we find indications for mutations of human Mut-S-Homologon-2 gene by immunostaining sporadic endometrial carcinomas using an antibody that is suggested to detect exclusively functionally active human Mut-S-Homologon-2 protein?
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Endometrial Specimens
Freshly excised endometrial specimens (malignancies of the uterine corpus, n = 40; normal endometrial tissue, n = 15) were immediately embedded in OCT Tissue-Tek II (Miles Scientific; Naperville, IL), snap-frozen in liquid nitrogen, and stored at -80C. Most of the neoplasms of the uterine corpus were sporadic endometrial adenocarcinomas (serous adenocarcinoma, n = 4; mucinous adenocarcinoma, n = 2; endometrioid adenocarcinoma, n = 28; clear-cell adenocarcinoma, n = 2). Four tumors were mixed epithelialnonepithelial tumors (heterologous carcinosarcomas, n = 4). All specimens of normal endometrial tissue were obtained from patients who underwent surgery for leiomyomas of the uterine corpus. All of these patients were in the secretory menstrual phase. The histopathological diagnosis of normal endometrial tissue was confirmed by a specialized histopathologist (K.R.).
Primary Antibody
Human Mut-S-Homologon-2 was detected by mouse monoclonal antibody FE11 (Dianova NA27; Hamburg, Germany). A polyclonal rabbit antibody directed against the Ki-67 antigen (Dako A 047; Hamburg, Germany) was used to immunophenotype proliferating cells in malignancies of the uterine corpus.
Preparation of Sections and Fixation
Serial sections (7 µm) were cut on a cryostat (ReichertJung; Heidelberg, Germany) and mounted on pretreated glass slides. Pretreatment of slides with 2% aminopropylmethoxysilane (Sigma; München, Germany) in acetone for 5 min was performed to enhance sticking of sections during the staining procedure. Sections to be stained for human Mut-S-Homologon-2 were fixed in 3.7% paraformaldehyde (Merck 4005; Darmstadt, Germany) in PBS [10 min at room temperature (RT)], incubated in methanol (Merck 6009; 3 min, -20C), and transferred into PBS. Sections to be stained for Ki-67 were air-dried (2 hr, RT), followed by fixation in acetone (10 min, RT), air-drying (a few minutes), chloroform (10 min, RT), air-drying, and rinsing in 0.05 M Tris-buffered saline (TBS), pH 7.6 (10 min, RT).
In Situ Detection of Human Mut-S-Homologon-2 Protein and Ki-67 Antigen
Incubation steps were performed in a moist chamber at RT, covering the sections with 100 µl of the respective reagents. The slides were incubated with the human Mut-S-Homologon-2 antibody at a dilution of 1:20 and with the Ki-67 antibody at a dilution of 1:200. After intermediate washing steps (PBS/TBS twice for 5 min), the sections were incubated with biotin-labeled rabbit anti-mouse IgG or goat anti-rabbit IgG (Dako) at a dilution of 1:400 (30 min, RT) and incubated with streptavidinperoxidase complexes (Dako; 1:400, 30 min, RT). After rinsing, the sections were incubated with 3-amino-9-ethylcarbazole (AEC; Sigma A5754) as a substrate for the peroxidase reaction, transferred into tapwater, and mounted with Aquatex (Merck).
In control sections, primary antibodies were replaced with polyclonal mouse IgG (Dako) or polyclonal rabbit IgG (Dako). No immunoreactivity was observed in control sections.
Semiquantitative Analysis of Immunoreactivity
Microscopic analysis was performed by three independent observers (M.F., K.R., and A.W.H.) including a specialized histopathologist (K.R.). Human Mut-S-Homologon-2 staining intensity (human Mut-S-Homologon-2-SI), percentage of human Mut-S-Homologon-2-positive cells (human Mut-S-Homologon-2-PP), and a resulting human Mut-S-Homologon-2 immunoreactivity score (human Mut-S-Homologon-2-IRS) were assessed as a modification of the technique described previously for estrogen and progesterone receptors (
Statistics
Statistics were performed by using the KruskalWallis and MannWhitney UWilcoxon Rank Sum W-tests. Statistical significance was defined as p<0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of Human Mut-S-Homologon-2 in Normal Endometrial Tissue
A total of 33% of normal endometrial tissues were human Mut-S-Homologon-2-negative, whereas the remaining 67% revealed weak human Mut-S-Homologon-2 immunoreactivity (mean human Mut-S-Homologon-2-IRS 1.25 ± 1.29; mean human Mut-S-Homologon-2-PP 8.92 ± 12.49; mean human Mut-S-Homologon-2-IS 0.83 ± 0.83). In the endometrial specimens, the connective tissue always gave negative staining results. Fibroblasts, identified by their shape, revealed either negative or very weak staining for human Mut-S-Homologon-2. Endothelial cells of capillaries, arterioles, and venules, as well as pericytes or smooth muscle cells in the microvascular wall, revealed either negative or very weak human Mut-S-Homologon-2-immunoreactivity as well (data not shown).
Expression of Human Mut-S-Homologon-2 in Malignancies of the Uterine Corpus
All analyzed malignancies of the uterine corpus revealed strong nuclear immunoreactivity for human Mut-S-Homologon-2 (mean human Mut-S-Homologon-2-IRS 9.00 ± 3.16; mean human Mut-S-Homologon-2-PP 79.38 ± 16.01; mean human Mut-S-Homologon-2-IS 2.50 ± 0.63). There was no visual difference in comparing the labeling pattern for human Mut-S-Homologon-2 in different types of malignancies of the uterine corpus. Tumor cells revealed consistently heterogeneous and strong human Mut-S-Homologon-2 immunoreactivity (Figure 1; Table 1). In addition, expression of human Mut-S-Homologon-2 protein was consistently and markedly upregulated in tumor cells of malignancies of the uterine corpus compared with normal endometrial tissue (Figure 1; Table 1). This upregulation was statistically significant (human Mut-S-Homologon-2-PP p<0.001; human Mut-S-Homologon-2-IS p<0.001; human Mut-S-Homologon-2-IRS p<0.001).
|
|
Comparison of Human Mut-S-Homologon-2 Expression to Expression of Ki-67 Antigen in Malignancies of the Uterine Corpus
All analyzed malignancies of the uterine corpus revealed weak to strong nuclear immunoreactivity for Ki-67 (mean percentage of Ki-67-positive tumor cells 22.00 ± 17.20). Most tumors revealed heterogeneous labeling for Ki-67, with no visual differences between central and peripheral tumor areas (Figure 1). There were no statistically significant correlations between the expression of Ki-67 and the expression of human Mut-S-Homologon-2 (human Mut-S-Homologon-2-PP p=0.443; human Mut-S-Homologon-2-IS p=0.234; human Mut-S-Homologon-2-IRS p=0.173).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To our knowledge, we have extensively analyzed for the first time the expression of the DNA mismatch repair enzyme human Mut-S-Homologon-2 in malignancies of the uterine corpus by immunohistochemistry. All endometrial carcinomas analyzed revealed strong nuclear immunoreactivity for human Mut-S-Homologon-2. In addition, our results revealed upregulation of human Mut-S-Homologon-2 protein in malignancies of the uterine corpus compared with normal endometrial tissue. It has previously been demonstrated that the majority of inactivating mutations of human Mut-S-Homologon-2 lead to a lack of expression or the expression of a truncated protein not detectable by the antibody that we used in this study (
Second, our results suggest that the expression of functionally active human Mut-S-Homologon-2 is upregulated in sporadic endometrial carcinomas. This human Mut-S-Homologon-2 upregulation may be caused by the neoplastic process driven by an increase in the rate of mutations in oncogenes and tumor suppressor genes. When we compared expression of human Mut-S-Homologon-2 protein with that of Ki-67 antigen in sequential sections, tumor specimens revealed no visual correlation. These findings suggest that expression of human Mut-S-Homologon-2 is not exclusively regulated by the proliferative activity of these tumor cells but instead is influenced by different unknown mechanisms. It can be speculated whether upregulation of human Mut-S-Homologon-2 in malignancies of the uterine corpus may be induced by other genes involved in postreplication recovery.
Recently, it has been shown that mutations in distinct mutS and mutL genes can block nucleotide excision repair, indicating a direct contribution of DNA mismatch repair enzymes to nucleotide excision repair pathways (
Because nucleotide excision repair removes damaged DNA (i.e., cross linked DNA after many anticancer treatments), upregulation of biologically and functionally active human Mut-S-Homologon-2 in malignancies of the uterine corpus may enhance DNA repair capacity and increase the resistance of tumor cells to radiotherapy or chemotherapy. Upregulation of functionally active human Mut-S-Homologon-2 may be a mechanism at least in part responsible for the lack of effectiveness of various chemotherapy regimens in sporadic endometrial carcinomas. The analysis of tumors for DNA repair capacity, including human Mut-S-Homologon-2 status, might be of value in predicting the therapeutic response to chemotherapy or radiation. Both the treatment and the prognosis of endometrial carcinoma are strongly related to the stage of the disease (5-year survival rate for Stage I endometrial carcinoma 8295%; for Stage II endometrial carcinoma 5060%; for Stage III endometrial carcinoma 1525%; for Stage IV endometrial carcinoma <10%) (
It was recently shown that the human Mut-S-Homologon-2 gene is directly regulated by p53 (
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berek JS, Hacker NF, Hatch KD, Young RC (1988) Uterine corpus and cervical cancer. Curr Prob Cancer 12:61-131[Medline]
Burks RT, Kessis TD, Cho KR, Hedrick L (1994) Microsatellite instability in endometrial carcinoma. Oncogene 9:1163-1166[Medline]
Creasman WT, Morrow CP, Bundy BN, Homesley HD, Graham JE, Heller PB (1987) Surgical pathologic spread patterns of endometrial cancer. A gynecologic oncology group study. Cancer 60(suppl 8):2035-2041[Medline]
Duggan DB, Felix JC, Muderspach LI, Tourgeman D, Zheng J, Shabita D (1994) Microsatellite instability in sporadic endometrial carcinoma. J Natl Cancer Inst 86:1216-1221[Abstract]
Fishel FS, Lescoe MK, Rao MR, Copeland NG, Jenkins NA, Garber J, Kane M, Kolodner R (1993) The human mutator gene homolog MSH-2 and its association with nonpolyposis colon cancer. Cell 75:1027-1038[Medline]
Grigsby PW, Perez CA, Kuske RR, Kao MS, Galakatos AE (1987) Results of therapy, analysis of failures, and prognostic factors for clinical and pathologic stage III adenocarcinoma of the endometrium. Gynecol Oncol 27:44-57[Medline]
Jovanovic AS, Boynton KA, Mutter GL (1996) Uteri of women with endometrial carcinoma contain a histopathological spectrum of monoclonal putative precancers, some with microsatellite instability. Cancer Res 56:1917-1921[Abstract]
Katabuchi H, van Rees B, Lambers AR, Ronnett BM, Blazes MS, Leach FS, Cho KR, Hedrick L (1995) Mutations in DNA mismatch repair genes are not responsible for microsatellite instability in most sporadic endometrial carcinomas. Cancer Res 55:5556-5560[Abstract]
Koreth J, O'Leary JJ, McGee O'DJ (1996) Microsatellites and PCR genomic analysis. J Pathol 178:239-248[Medline]
Mellon I, Champe GN (1996) Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide excision repair of the lactose operon Escherichia coli. Proc Natl Acad Sci USA 93:1292-1297
Mellon I, Rajpal KD, Koi M, Boland RC, Champe GN (1996) Transcription-coupled repair deficiency and mutations in human mismatch repair genes. Science 272:557-560[Abstract]
Papadopoulos N, Nicolaides NC, Liu B, Parsons RE, Lengauer C, Palomobo F, Darrigo A, Markowitz S, Willson JKV, Kinzler KW, Jiricny J, Vogelstein B (1995) Mutations of GTBP in genetically unstable cells. Science 268:1915-1917[Medline]
Prolla TA, Abuin A, Bradly A (1996) DNA mismatch repair deficient mice in cancer research. Cancer Biol 7:241-247
Quinn AG, Healy E, Rehman I, Sikkink S, Rees JL (1995) Microsatellite instability in human non-melanoma and melanoma skin cancer. J Invest Dermatol 104:309-312[Abstract]
Remmele W, Hildebrand U, Hienz HA, Klein PJ, Vierbuchen M, Behnken LJ, Heicke B, Scheidt E (1986) Comparative histological, histochemical, immunhistochemical and biochemical studies on estrogen receptors, lectin receptors, and Barr bodies in human breast cancer. Virchows Arch 409:127-147
Risinger JI, Berchuk A, Kohler MF, Watson P, Lynch HT, Boyd J (1993) Genetic instability of microsatellites in endometrial carcinoma. Cancer Res 53:1-4[Abstract]
Scherer JS, Welter C, Zang KD, Dooley S (1996) Specific in vitro binding of p53 to the promotor region of the human mismatch repair gene human Mut-S-Homologon-2. Biochem Biophys Res Commun 221:722-778[Medline]
Taghian A, Pernot M, Hoffstetter S, Luporosi E, Bey P (1988) Radiation therapy alone for medically inoperable patients with adenocarcinoma of the endometrium. Int J Radiat Oncol Biol Phys 15:1135-1140[Medline]