Nucleolar organizer regions in various human brain tumors

Size: px

Start display at page:

Download "Nucleolar organizer regions in various human brain tumors"

Ashley Carter
5 years ago
Views:

J Neurosurg 74:979-984, 1991 Nucleolar organizer regions in various human brain tumors TETSUYA SHIRAISHI, M.D., KAZUO TABUCHI, M.D., TOSHIHIRO MINETA, M.D., NOBUAKI MOMOZAKI, M.D., AND MASASHI TAKAGI, M.

1 J Neurosurg 74: , 1991 Nucleolar organizer regions in various human brain tumors TETSUYA SHIRAISHI, M.D., KAZUO TABUCHI, M.D., TOSHIHIRO MINETA, M.D., NOBUAKI MOMOZAKI, M.D., AND MASASHI TAKAGI, M.D. Department of Neurosurgery, Saga Medical School, Saga, Japan t,- Nucleolar organizer regions (NOR's) are loops of deoxyribonucleic acid (DNA) which transcribe to ribosomal ribonucleic acid (RNA) by RNA polymerase I. They possess vital significance in the ultimate synthesis of cellular proteins. A silver colloid staining technique for demonstration of NOR-associated proteins (Ag-NOR's) was applied to paraffin-embedded sections from 128 varied brain tumors and to chromosomal preparations from cultured brain-tumor cells. There was a statistically significant difference in the mean number of Ag-NOR's per nucleus between low-grade tumors (l.98/nucleus) and high-grade tumors (2.95/ nucleus). It is suggested that the mean number of Ag-NOR's may represent the proliferative potential of brain tumors. Furthermore, high-grade tumors usually showed relatively large Ag-NOR's in a scattered distribution. In chromosomal preparations, the cultured cells displayed five to 12 Ag-NOR's on acrocentric chromosomes. Five of eight cell lines examined demonstrated ectopic Ag-NOR's. This simple staining technique can be easily applied to routinely processed paraffin-embedded sections and will become a useful tool for quick estimation of the proliferative potential of human brain tumors. KEy WORDS 9 nucleolar organizer region 9 silver colloid staining 9 brain tumor 9 proliferative potential 9 chromosome A VARIETY of histological procedures have been applied to biopsy specimens of brain tumors in an effort to evaluate their proliferative activity. Recently, immunohistochemical assessment of proliferating cells has been attempted using antibodies to bromodeoxyuridine (BUdR) ~8 and proliferating cell nuclear antigen (PCNA/cyclin), 2s and Ki-67 antigen. 2~ The BUdR method requires preoperative injection of BUdR, which may be toxic to the patient. Since PCNA/ cyclin and Ki-67 antigen easily lose their antigenic activities in conventional fixation procedures, 26 immunohistochemical staining with these antibodies is not suitable for retrospective studies on stored formalinfixed paraffin-embedded tissue specimens. Nucleolar organizer regions (NOR's) have been shown to be loops of ribosomal deoxyribonucleic acid (rdna) which transcribe to ribosomal ribonucleic acid (rrna) ~ and are associated with several proteins. Since NOR-associated proteins (Ag-NOR's) are argyrophilic, silver staining methods have been used for demonstration of Ag-NOR's in chromosomal preparation. In 1980, Howell and Black ~9 developed a simple one-step silver colloid staining method which can clearly identify NOR's as black dots on paraffin sections under a pho- tomicroscope, It has been suggested that the number and size of Ag-NOR's correlate with cellular activity in general and may be an indicator of the degree of proliferative potential or malignancy of human neoplasms?,5.9-~ 1,14 We have examined the number and size of Ag-NOR's and have correlated their distribution pattern in paraffin-embedded tissue samples of various human brain tumors with their histological grades of malignancy according to the World Health Organization grading system. 3~ Nucleolar organizer regions are located on the five acrocentric chromosome pairs; 2 however, they have been reported in abnormal locations (ectopic NOR's) in human testicular tumors. 7 We also examined the expression of NOR's in the chromosomes of cultured cells from human brain tumors. Materials and Methods Staining of Ag-NOR's The tumor specimens examined were obtained from 128 varied brain tumors during surgical operations. Specimens were routinely fixed in 10% neutral buffered formalin and processed with paraffin wax. Sections of J. Neurosurg. / Volume 74/June,

T. Shiraishi, et al. F](~. 1. L~{~:Photomicrograph of a high-grade astrocytoma demonstrating a mean number of nucleolar organizer region-associatedproteins (Ag-NOR's)of 2.6 per nucleus.

10 ~m were cut and dewaxed in xylene, and then hydrated through decreasing grades of ethanol to deionized distilled water.

2 T. Shiraishi, et al. F](~. 1. L~{~:Photomicrograph of a high-grade astrocytoma demonstrating a mean number of nucleolar organizer region-associatedproteins (Ag-NOR's)of 2.6 per nucleus. Two or three distinct black dols are present within each nuclei. Ag-NORstain, x 285. Right: Photomicrographof a medul[oblastomashowing increased number of small Ag-NOR's.Ag-NOR slain, x ~m were cut and dewaxed in xylene, and then hydrated through decreasing grades of ethanol to deionized distilled water. The Ag-NOR staining was carried out by the one-step method of Howell and Black.~9 Briefly, a colloidal developer solution (freshly made each time), consisting of one volume of 2% gelatin in 1% aqueous formic acid and two volumes of 50% aqueous silver nitrate solution, was reacted on the surface of the sections for 60 minutes under safelight conditions at room temperature. The sections were washed with deionized water and dehydrated in ascending ethanol concentrations. The sections were placed in xylene and mounted in a synthetic medium. No counterstain was applied. Cell Culture The biopsy specimens were minced into pieces approximately 1 mm in diameter. The fragments were then layered on the bottom of Falcon plastic flasks with 5 ml of medium. To initiate the culture, Dulbecco's modified Eagle's medium (MEM), supplemented with % inactivated fetal bovine serum (FBS) containing penicillin G (0 IU/ml), was used. All cultures were incubated at 37"C in a humidified 5% CO2 atmosphere. When the primary culture reached confluence, the cells were transferred by treatment with 0.05% trypsin and 0.02% ethylenediaminetetra-acetic acid, and maintained in the same medium but containing 10% FBS. Established cultured glioma cell lines were maintained using the same procedure. Evaluation of Ag-NOR "s In each case, about 0 nuclei were examined under a x 100 oil-immersion objective lens. Multiple regions from each section were examined. The silver-stained dots were counted and the mean number of Ag-NOR's was determined for each nucleus. Careful focusing was used to visualize all Ag-NOR's in a nucleus. The areas 980 of the nucleoli and the Ag-NOR's of each cell were also measured by means of a microcomputer-assisted image analysis system, and the percentage of mean Ag-NOR area to mean nucleolus area was calculated (%A/N). The differences between the groups were analyzed by the use of Student's t-test. Chromosome Analysis To obtain cells in metaphase from established glioma cell lines and primary cultured tumor cells, cells were incubated in MEM containing 0.5 #g/ml of Colcemid for 6 hours. After hypotonic treatment (0.56% KCI) and triple fixation (3:1 methanol:acetic acid), slides were made and flame-dried. The Ag-NOR's were stained on the slides, and the slides were then rinsed several times in distilled water and counterstained with Giemsa solution for 2 to 3 minutes. Results Biopsy Specimens In all specimens, a variable number of silver dots (Ag-NOR's) were clearly stained black in yellow-stained nuclei. In seven cases of low-grade astrocytomas (grade 1 or 2), the mean number of Ag-NOR's per nucleus was 1.98 _ (_+ standard error of the mean). The mean nucleolus area, Ag-NOR area, and %A/N for these cells were.1 sq urn, 1.3 sq urn, and 6.5%, respectively. The Ag-NOR's were similar in size and location near the periphery, of the nuclei. Twenty-seven cases of high-grade astrocytomas (grade 3 or 4) showed a higher mean number of Ag-NOR's (2.41 _+0.39) than that of low-grade astrocytomas. The mean of %A/N was increased as well, to 11.1%. These tumors also displayed moderate pleomorphism of Ag-NOR's (Fig. I left). The highest mean number (3.57 _+ 0.88) of AgNOR's per nucleus among the brain tumors were found in two cases of medulloblastomas; however, the Ag- J. Neurosurg. / Volume 74/June. 1991

Ag-NOR stain, x 280. FJ(~. 3. Photomicrograph of a malignant lymphoma demonstrating a mean number of nudeolar organizer regionassociated proteins (Ag-NOR's) of 3.8 per nucleus. Ag-NOR stain, x 285.

3 Nucleolar organizer regions in brain tumors HG. 2. Photomicrograph of a pituilary adenoma showing large nucleolar organizer region-associated protein (Ag-NOR) dots located in the central portion and smaller Ag-NOR dots in the periphery of the nuclei. Ag-NOR stain, x 280. FJ(~. 3. Photomicrograph of a malignant lymphoma demonstrating a mean number of nudeolar organizer regionassociated proteins (Ag-NOR's) of 3.8 per nucleus. Ag-NOR stain, x 285. NOR's were generally small (%A/N 5.6%) and were scattered throughout the nucleus (Fig. 1 righo. Among 53 cases of meningiomas there were a few large Ag-NOR's (mean 1.97 _+ 0.38), which were in a clustered distribution. Four cases of malignant meningiomas possessed a significantly high mean number of Ag-NOR's (3.22 _+ 0.68). The mean %A/N of meningiomas and malignant meningiomas was 8.0% and 10.8%, respectively. The mean Ag-NOR was 1.89 _ 0.28 (%A/N 7.4%) for 11 cases of neurinoma and 2.06 _ for seven cases of pituitary adenoma. There was a tendency for larger Ag-NOR's to be found in the central area and smaller ones at the periphery (Fig. 2). Macroadenomas had a higher mean number of Ag- NOR's ( , three cases) than did microadenomas (1.72 _+ 0.39, four cases). Three cases of craniopharyngiomas had a relatively high mean number of Ag-NOR's (2.36 _+ 0.28) and %A/N (12.9%) compared to other benign tumors. This might reflect that craniopharyngiomas tend to have increased activity of secretion compared to other benign tumors. Four cases of malignant lymphomas revealed many Ag-NOR's (mean number ) with a large area (%A/N 15.5%) and with a highly irregular shape (Fig. 3). These results are expressed as a scattergram in Fig. 4. There was a statistically significant difference in the mean number of Ag-NOR's in the nucleus between the 81 benign tumors (Grades I and 2: 1.98/nucleus) and the 47 malignant tumors (Grades 3 and 4: 2.95/nucleus; p < 0.01). Morphometric findings demonstrated a generally increasing trend in the areas of the Ag-NOR's with increasing tumor malignancy. However, it is noteworthy that medulloblastomas have small Ag-NOR's in spite of being malignant tumors and craniopharyngiomas have large Ag-NOR's in spite of being benign tumors. FIG. 4. Scattergram showing nucleolar organizer regionassociated protein (Ag-NOR) numbers per nucleus for various brain tumors. ~77romosome Analysis In chromosomal preparations, all cultured cells except T-98G expressed between five and eight Ag-NOR's on acrocentric chromosomes. The T-98G glioma cells expressed 12 Ag-NOR's and three constant ectopic Ag- Z ~k, urosurg. / Volume 74/June,

4 T. Shiraishi, et al. FIG. 5. Si ver stainedhumanmetaphasechr m s mesfr mthet-98gg i mace ine.nuc e ar rganizer region-associated proteins (Ag-NOR's) appear as black dots above the satellite stalks o f short arms of the acrocentric chromosomes. Arrows indicate ectopic Ag-NOR's. Ag-NOR and Giemsa stain, 830. a fibrillar center, a dense fibrillar component, and a granular component.~ Among these three components, the fibrillar centers are the so-called "nucleolar organizer regions" which consists of loops of DNA transcribing to 18S and 28S rrna subunits. ~'~2Although activated NOR's are found in the nucleolus, inactive NOR's may be detected in the extranucleolar nucleoplasm. 29 Several proteins, including RNA polymerase I, 24 C2s protein (nucleolin), 22 B23 protein, 2j 100-kDa proteinff and 80-kDa protein, 3 are known to be associated with NOR's. They include carboxyl- and sulfur-containing proteins that are of essential importance in the Ag- NOR's. These ectopic sites were located near the centromere of A, B, and C group-sized chromosomes. Abstracts of karyotypes are shown in Fig. 5 and the number of Ag-NOR's on chromosomes in certain cultured brain cells is shown in Table 1. Discussion Nucleolar OrganizerRegions The nucleolus is known to be a subce]lular organelle producing rrna and to be composed of the three components discernible under an electron microscope: TABLE 1 Ag-NOR "s on chromosomes of cultured brain tumor cells* Cell Line Origin of Cells No. of Cells Counted Modal No. of Chromosomes A 172 T-98G U251 ME-1 ME-2 ME-3 ME-4 GM-l glioblastoma glioblastoma mixed glioma glioblastoma, primary culture Modal No. of D+G Group Modal No. of Ag-NOR's Ectopic Ag-NOR's yes (B,E), often yes (A,B,C), constant no no no yes (B), often yes (B), often yes (A), often * A g - N O R ' s = nucleolar organizer region-associated proteins; A. B, C, E = A, B, C, E group-sized chromosomes. 982 J. Neurosurg. / Volume 74/June, 1991

Nucleolar organizer regions in brain tumors NOR reaction? 7 They are thought to act as regulators of DNA at NOR transcription ~ or to maintain the extended configuration of DNA at NOR's.

5 Nucleolar organizer regions in brain tumors NOR reaction? 7 They are thought to act as regulators of DNA at NOR transcription ~ or to maintain the extended configuration of DNA at NOR's. t7 Corre]alion Belweetl &g-nor's and Mal(~,namT The exact significance of changes in the number and distribution of Ag-NOR's is not fully understood. However, since NOR's are transcribed to rrna and thus ultimately to ribosomes and finally to protein, it has been speculated that an increased number of Ag-NOR's might reflect an increased rdna transcriptional activity or potential ~ and might indicate increased nucleolar and cellular activities) ~3 Therefore, Ag-NOR's have been used as an index for the proliferative activity of cells. There have been several reports that higher numbers of Ag-NOR's correlate well with high-grade malignancy in intestinal tumorsfl neuroblastomas, ~~ malignant melanocytic lesions," and non-hodgkin's lymphomas? Hall, el al., ~4 reported that the proliferative activity of tumors, as determined by immunostaining with the monoclonal antibody Ki-67 (a marker of cell proliferation), correlated well with the mean number of Ag-NOR's in the tumor cells. Correlation was also reported between the mean number of Ag- NOR's and the S-phase cell populations determined by BUdR immunohistochemistry 2~' or DNA flow cytometry. 4 These data suggest that the mean number of Ag- NOR's may reflect the cellular proliferative activity. The authors studied a total of 128 human brain tumors in relation to the number and size of Ag-NOR's as well as their distribution pattern. A statistically significant difference was observed in the mean number of Ag-NOR's in the nuclei between low-grade tumors and high-grade tumors. There was also a tendency that the size of Ag-NOR's in high-grade tumors was increased compared to that in low-grade tumors. Cells of low-grade tumors tend to have a regular nucleolus with tightly clustered Ag-NOR's, while high-grade tumor cells often show dispersed Ag-NOR's throughout the nucleus assvnj asmultiple nucleoli containing clustered Ag-NOR's. Ag-NOR '6 on Chromosomes Nucleolar organizer regions are located on the satellite stalks of short arms of the five acrocentric chromosome pairs (Nos. 13, 14, 15, 21, and 22) in human diploid cells. ~L5 The number of Ag-NOR's on chromosomes in human diploid cells varies from five to 10, with a mean of seven to eight in phytohemagglutininstimulated lymphocytes. -'s Cytogenic studies have shown ectopic Ag-NOR's or unusual Ag-NOR patterns in certain malignancies. 7-'~ Our findings revealed that cultured brain-tumor cells except T-98G expressed five to eight Ag-NOR's on acrocentric chromosomes; the T- 98G glioma cells possessed an increased number of Ag- NOR's on chromosomes. Except for T-98G cells, the number of Ag-NOR's was similar to that reported in normal cells in spite of their increased modal chromo- some number and increased number of acrocentric chromosomes. Five of eight cell lines demonstrated inconstant ectopic Ag-NOR's. The T-98G glioma cells have three constant ectopic Ag-NOR's on their chromosomes and their increased Ag-NOR's may be ascribed to these ectopic Ag-NOR's. The active ectopic NOR's may be due to the participation of short-arm acrocentric chromosomes in translations or derepression of previously existing but inactive NOR sites on nonacrocentric chromosomes] It will be worthwhile to investigate the biological role of ectopic NOR's in brain tumors. Conclusions Silver colloid staining seems to be a useful method for evaluation of proliferative activity of brain tumors. It has an advantage over immunohistochemical methods because it can be easily applied to conventionally fixed and processed paraffin sections and enables the retrospective study of brain-tumor specimens for their proliferative potential. Acknowledgment We wish to thank Miss Yumiko Saho for technical assistance. References 1. Alberts B, Bray D, Lewis J, et al: The cell nucleus, in Alberts B. Bray D, Lewis J, et al (eds): Molecular Biology of the Cell, ed 2. New York: Garland Publishing, 1989, pp Cheung SW, Sun L, Featherstone T: Visualization of NORs in relation to the precise chromosomal localization of ribosomal RNA genes. Cytogenet Cell Genet 50: 93-97, Courvalin JC, Maunoury R, Hernandez-Verdun D, et al: Une proteine de 80kD esl associee a l'organisateur nucleolaire (NOR) des cellules humaines. Biol Cell 49:10a, 1983 (Abstract) 4. Crocker J, Macartney JC, Smith P J: Correlation between DNA flow cytometric and nucleolar organizer region data in non-hodgkin's lymphomas. J Pathol 154: , Crocker J, Nat P: Nucleolar organizer regions in lymphomas. J Pathol 151: , Das BC, Rant R, Mitra AB, et al: The number of silverstaining NORs (rdna) in lymphocytes of newborns and its relationship to human development. Mech Ageing Dev 36: , DeLozier-Blanchet CD, Walt H, Engel E: Eetopic nucleolus organizer regions (NORs) in human testieular tumors. Cytogenet Cell Genet 41: , Derenzini M, Farabegoli F, Pession A, et al: Spatial redistribution of ribosomal chromatin in the fibrillar centres of human circulating lymphocytes after stimulation of transcription. Exp Cell Res 170:31-4l, Derenzini M, Romagnoli T, Mingazzini P, et al: Interphasic nucleolar organizer region distribution as a diagnostic parameter to differentiate benign from malignant epithelial tumors of human intestine. Virch Arch (B) 54: , Egan M, Raafat F, Crocker J, eta[: Comparative study of" J. Neurosurg. / Volume 74/June, 199I 983

6 T. Shiraishi, et al. the degree of differentiation of neuroblastoma and mean numbers of nucleolar organiser regions. J Clin Pathol 41: , Egan M J, Crocker J: Nucleolar organizer regions in cutaneous tumours. J Pathol 154: , Fakan S, Hernandez-Verdun D: The nucleolus and the nucleolar organizer regions. Biol Cell 56:189-6, Field DH, Fitzgerald PH, Sin FYT: Nucleolar silverstaining patterns related to cell cycle phase and cell generation of PHA-stimulated human lymphocytes. Cytohios 41:23-33, Hall PA, Crocker J, Watts A, et al: A comparison of nucleolar organizer region staining and Ki-67 immunostaining in non-hodgkin's lymphoma. Histopathology 12: , Henderson AA, Warburton D, Atwood KC: Location of ribosomal DNA in the human chromosome complement. Proc Natl Acad Sci USA 69: , Hernandez-Verdun D: The nucleolar organizer regions. Biol Cell 49:191-2, Hernandez-Verdun D, Derenzini M, Bouteille M: Relationship between AgNOR proteins and ribosomal chromatin in situ during induced RNA synthesis inhibition. J Ultrastruct Res 88:55-65, Hoshino T, Nagashima T, Murovic JA, el al: In situ cell kinetics studies on human neuroectodermal tumors with bromodeoxyuridine labeling. J Neurosurg 64: , Howell WM, Black DA: Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a l-step method. Experientia 36: , Hubbell HR, Hsu TC: Identification of nucleolus organizer regions (NORs) in normal and neoplastic human cells by the silver-staining technique. Cytogenet Cell Genet 19: , Lischwe MA, Smetana K, Olson MOJ, et al: Proteins C23 and B23 are the major nucleolar silver staining proteins. Life Sci 25: , Ochs RL, Busch H: Further evidence that phosphoprotein C23 (110 kd/pi 5.1) is the nucleolar silver staining protein. Exp Cell Res 152: , Olson MOJ, Thompson BA: Distribution of proteins among chromatin components of nucleoli. Biochemistry 22: , Scheer U, Rose KM: Localization of RNA polymerase 1 in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry. Proc Natl Acad Sci USA 81: , Schwarzacher HG, Wachtler F: Nucleolus organizer regions and nucleoli. Hum Genet 63:89-99, Shiraishi T: Cell kinetic analysis of brain tumors using the monoclonal antibody Ki-67: in vitro and in situ study. Acta Med Okayama 43:187-1, Smith PJ, Skilbeck N, Harrison A, et al: The effect of a series of fixatives on the AgNOR technique. J Patho1155: , Tabuchi K, Honda C, Nakane PK: Demonstration of proliferating cell nuclear antigen (PCNA/cyclin) in glioma cells. Neurol Med Chir 27:1-5, Tanaka T, Takeuchi T, Nishikawa A, et al: Nucleolar organizer regions in hepatocarcinogenesis induced by N- 2-Fluorenylacetamide in rats: comparison with bromodeoxyuridine immunohistochemistry, dpn J Cancer Res 80: , , Ztilch KJ: Histological typing of tumors of the central nervous system, in: International Histological Classification of Tumours, No. 21. Geneva: World Health Organization, 1979 Manuscript received March 15, Accepted in final form October 25, Address reprint requests to." Tetsuya Shiraishi, M.D., Department of Neurosurgery., Saga Medical School, Nabeshima, Saga 849, Japan. 984 J. Neurosurg. / Volume 74~June, I991

Citation Acta medica Nagasakiensia. 1994, 39

NAOSITE: Nagasaki University's Ac Title Significance of AgNORs Ratio as a P Author(s) Hatano, Kazuhiko Citation Acta medica Nagasakiensia. 1994, 39 Issue Date 1994-10-25 URL http://hdl.handle.net/10069/15998