GYNECOLWIC
ONCOLOGY
39, 309-313 (1990)
Nucleolar Organizer Regions in Malignant Transformation of Uterine Cervix YASUHIRO YOKOYAMA,
M.D.,’
DILBAZ
SERDAR, M.D.,
KENNJI NIWA,
M.D.,
AND TERUHIKO
TAMAYA,
M.D.
Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu University, Gifu City, Japan Received March 22, 1990
Nucleolarorganizerregions(NORs) in 70 casesof precancerous and cancerouslesionsof the uterine cervix wereinvestigated.The greater the degreeof dysplasia,the higher the meannumber of silver-stainedNOR (Ag-NOR) dots (mild dysplasia,2.2; moderate dysplasia,2.5; severedysplasia,3.1). The meannumberof dots in moderateor severedysplasiawassignificantly higher than that in normal squamousepithelial cells. The mean numbersof dots in carcinomain situ (CIS) and invasive carcinomawere 3.8 and 3.7, respectively, and weresignificantly higherthan thosein normal squamous epithellalcells,mild dysplasia,moderatedysplasia, and severedysplasia.The meannumber of Ag-NOR dots in the casesof mild dysplasiathat progressedto CIS was 2.7, which wassignlflcantly higher than that in the casesthat regressed.In precancerouslesions,the correlationcoefficientbetweenthe mean number of Ag-NOR dots and the mitotic index was 0.552 (P < O.Ol), whereasno correlation wasnoted betweenthesemeasurementsin CIS and invasive carcinoma.In conclusion,the number of Ag-NOR dots is a good marker to detect cell proliferation in Aeadtic precancerouslesionsand their maliguanttendency. 9 1990 press, Inc.
INTRODUCTION
Nucleolar organizer regions (NORs) are specific sites of genes involved in the formation of the nucleolus in the human being, and are localized in the second constrictions of chromosomes 13, 14, 15, 21, and 22. The NORs can be demonstrated by silver staining, which is thought to visualize the acid proteins surrounding these genes [ 11. Silver-stained NORs (Ag-NORs) observed in the interphase of the cell cycle are ordinarily localized in nucleoli and are considered to contribute to the transcription and/or maturation of rRNA [2]. The intensity of silver staining is thought to have relevance to the I To whom correspondence Gifu City, 500, Japan.
should be sent at Tsukasa-machi
40,
transcriptional activity or potential of such genes [3] and to the progress of the malignant transformation [4,5]. Squamous cell carcinoma of the uterine cervix is speculated to originate in reserve cell proliferation, to which neoplastic initiation might be brought by unknown causes. The present study was designed to investigate quantitative changes in the Ag-NORs of squamous cells in the uterine cervix as a marker of malignant transformation. MATERIALS
AND METHODS
Seventy neoplastic lesions of the uterine cervix comprising 18 cases of mild dysplasia, 12 cases of moderate dysplasia, 17 cases of severe dysplasia, 11 cases of carcinoma in situ (CIS), 12 cases of invasive carcinoma, and 18 cases of normal uterine cervix (9 cases of normal cervical epithelium and 9 cases of squamous metaplasia) were studied. Mild, moderate, and severe dysplasia were defined as follows: mild, neoplastic cells within the lowest third of the epithelium; moderate, into the middle third; and severe, into the upper third. The samples were surgically resected or biopsied specimens obtained in Gifu University Hospital in the past 10 years. They were fixed in 10% neutral formalin and embedded in paraffin. Specimens were selected randomly with the exception of 5 cases of mild dysplasia, 5 cases of moderate dysplasia, and 9 cases of severe dysplasia that had subsequently progressed to carcinoma in situ. Two sequential 3-pm-thick sections were cut and deparaffinized. One section was used for Ag-NOR staining, the other for hematoxylin and eosin (HE) staining. The HE sections were examined to confirm the diagnosis and the mitotic index per 1000 neoplastic cells evaluated. The procedure for Ag-NOR staining was as follows: a solution, made up of 2% gelatin in 1% aqueous formic acid, was mixed with a twofold volume of 50% aqueous
309 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
FIG. 1.
Normal
FIG. 2. Moderate dysplasia. Ag-NOR dots. x530.
epithelium.
The basal and parabasal cells contain distinct and relatively
The dysplastic
cells have distinct Ag-NORs, 310
large Ag-NORs.
x530.
but the mature cells in the upper layer do not contain identifiable
NUCLEOLAR
FIG.
3.
ORGANIZER
REGIONS
IN UTERINE
Carcinoma in situ. The neoplastic cells contain increased numbers of small Ag-NOR dots.
silver nitrate. The mixture was poured on the sections, and incubated at room temperature for 30 min. The staining reaction was terminated to submerge the sections in running tap water. Stained sections were examined under an oil immersion lens at a magnification of 1000, and over 200 nuclei were studied. Cells with nuclear pyknosis were not evaluated because of the unreliability of Ag-NOR staining. Ag-NOR dots in each nucleus were counted, and the
311
CERVIX
X
1060.
mean number of Ag-NOR dots per nucleus was calculated for each section. Data obtained were evaluated by Student’s t test. RESULTS In normal squamous epithelium, the Ag-NORs were clearly observed only in cells of the basal and parabasal layers (Fig. 1); the cells in the superhcial layer did not
312
YOKAYAMA
ET AL.
AgW3?s Precancerous
lesion
(/Cell)
51
-
. +2.07442
i J0 .
0
IO 20 30 40 50 60 70 Mitotic
index
(/lOOOcells)
Carcinoma
Ag-NORs
(/Cell)
61
;I . . , , . ;=,,,,6 0 cases
whose
lesions
have
progressed
to CIS afterward
FIG. 4. Mean numbers of A&NOR dots per nucleus of specimens examined.
show distinct Ag-NOR dots because of pyknosis. The neoplastic cells in moderate dysplasia showed clear and relatively large Ag-NOR dots (Fig. 2), but cells that had matured in the upper layer did not. Carcinoma cells generally had small but increased numbers of Ag-NOR dots (Fig. 3). The mean number of Ag-NOR dots for each diagnostic category is shown in Fig. 4. In normal squamous epithelial cells and metaplastic squamous cells, the mean (&SD) numbers were 2.0 rfr 0.3 and 1.7 2 0.2, respectively. The more advanced the degree of atypia, the greater the mean number of Ag-NOR dots (mild dysplasia, 2.2 + 0.6; moderate dysplasia, 2.5 + 0.4; and severe dysplasia, 3.1 + 0.7). The numbers of Ag-NOR dots in moderate dysplasia (P < 0.01) and severe dysplasia (P < 0.001) were significantly higher than the number in normal squamous epithelial cells. The mean numbers of AgNOR dots in CIS and invasive carcinoma were 3.8 + 0.9 and 3.7 + 0.9, respectively. These numbers were significantly highr than those in normal squamous epithelial cells (P < O.OOl), mild dysplasia (P < O.OOl), moderate dysplasia (P < O.Ol), and severe dysplasia (P < 0.05). However, the difference between the numbers in CIS and invasive carcinoma was not significant. The mean number of Ag-NORs in the cases of mild dysplasia who progressed to ClS was 2.7 + 0.2, which was significantly higher (P < 0.05) than that in the cases that regressed (2.0 ? 0.6) (Fig. 4). As for moderate and
0 10 20 30 40 50 60 70 80 Mitotic index (/lo00 cells)
FIG. 5. Relationship between mitotic index and mean number of Ag-NORs in precancerous lesions and carcinomas.
severe dysplasia, there were no significant differences in the mean numbers of Ag-NOR dots between cases that progressed to CIS (2.6 2 0.3 and 3.2 + 0.6, respectively) and those that did not (2.4 f 0.4 and 3.0 + 0.8, respectively). The relationship between number of Ag-NOR dots and mitotic index in the precancerous lesions and in carcinoma is shown in Fig. 5. The correlation coefficient between these measurements in precancerous lesions was 0.552 (P < O.Ol), whereas no such correlation was recognized in cases of CIS and invasive carcinoma. DISCUSSION The nucleolus is a cellular factory that produces rRNA. Both the number and the size of the nucleoli are related to cellular activity. The genes that are involved in the formation of the nucleolus are localized in specific sites of defined chromosomes as observed during metaphase. With the progression of the cell cycle, the nucleolus can be formed around such genes (rDNA). These genes were first visualized by Goodpasture et al. [ 11 over a decade ago and the methodology subsequently became more stable and reliable [6]. The substance stained by the silver method is not considered to be the rDNA gene itself, but to consist of nonhistone proteins that exist in the vicinity of rDNA
NUCLEOLAR
ORGANIZER
REGIONS IN UTERINE
and play a role in the production and/or maturation of rRNA [7]. Lischwe et al. [8] proved by two-dimensional polyacrylamide gel electrophoresis that the major substances stained by silver in the Novikoff hepatoma ascites cell are proteins B23 and C23. Electron microscopic examination demonstrates that Ag-NOR dots are localized in fibrillar centers and around dense granular components [9-l I]. Cytochemical investigations show that fibrillar centers contain rDNA and numerous proteins (Ag-NOR proteins, RNA polymerase I, and C23 protein) and that dense fibrillar components contain pre-rRNP complexes and Ag-NOR proteins [ 121. The silver stainability may be considered a marker of both rDNA transcription activity and/or transcription potential. Cracker and Nar [ 131 reported that the number of Ag-NORs in high-grade lymphomas was significantly higher than that in low-grade lymphomas. Likovsky et al. [14] reported that the number of Ag-NORs in hepatoma cells was significantly higher than that in normal hepatocytes. Those results suggest that the number of Ag-NORs is related to neoplastic transformation and malignant potential in some species of cells. Uterine cervical squamous cell carcinomas develop in the transformation zone where squamous metaplasia frequently occurs. The metaplasia is hypothesized to result from proliferation and squamous metaplasia of reserve cells. The initiation of the transformation of reserve cells to precancerous lesions or carcinoma remains to be clarified. Of great importance is the discrimination between metaplastic cells and neoplastic cells. With silver staining, the metaplastic cells have a relatively small number of Ag-NOR dots. In contrast, in the intraepithelial neoplastic lesions, the more severe the dysplastic change, the greater the mean number of Ag-NORs dot is. These results are in agreement with those of Rowlands [4] and Egan et al. [5]. However, once the lesion progresses to carcinoma, the mean number of Ag-NORs does not change. In addition, the mean number of Ag-NORs dots in mildly dysplastic lesions that progress to CIS is higher than in those that regress. These results suggest that the number of Ag-NORs dots is related to the malignant potential of intraepithelial neoplasia. In intraepithelial neoplasia, the number of Ag-NOR dots is positively correlated to the mitotic index. How-
CERVIX
313
ever, this is not true in carcinomas. The former finding suggests that the number of Ag-NOR dots is also related to proliferative activity in intraepithelial neoplasia. ACKNOWLEDGMENTS We are very grateful to Associate Professor Shimokawa (Department of Central Laboratory, Gifu University Hospital) and Professor Mori (Department of 1st Pathology, Gifu University School of Medicine) for their contributions to this study.
REFERENCES 1. Goodpasture, C., and Bloom, S. E. Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining, Chromosoma (Berlin) 53, 37 (1975). 2. Carneiro, V., Deltour, R., Fakan, S., et al. The nucleolus and the nucleolar organizer regions, Viol. Cell. 56, 189 (1986). 3. Hofgirtner, F. J., Krone, W., and Jain, K. Correlated inhibition of ribosomal RNA synthesis and silver staining by actinomycin D, Hum. Genet. 47, 329 (1979). 4. Rowlands, D. C. Nucleolar organizer regions in cervical intraepithelial neoplasia, J. Clin. Pathol. 41, 1200 (1988). 5. Egan, M., Freeth, M., and Cracker, J. Relationship between intraepithelial neoplasia of the cervix and the size and number of nucleolar organizer rkgions, Gynecol. Oncol. 36, 30 (1990). 6. Howell, W. M., and Black, D. A. Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: A l-step method, Experientiu 36, 1014 (1980). 7. Hernandez-Verdum, D. (Ed.). The nucleolar organizer regions (collective review), Biol. Cell. 49, I91 (1983). 8. Lischwe, M. A., Smetana, K., Olson, M. 0. J., and Busch, H. Proteins C23 and B23 are the major nucleolar silver staining proteins, Life Sci. 25, 701 (1979). 9. Schwarzacher, H. G., Mikelsaar, A. V., and Schnedl, W. The nature of the nucleolus organizer regions: Electron- and light-microscopic studies on human cells in interphase, mitosis, and meiosis, Cytogenet. Cell Genet. 20, 24 (1978). 10. Goessens, G. Localisation of nucleolus-organising regions in interphase cells, Cell Tissue Res. 200, 159 (1979). Il. Bourgeois, C. A., Hernandez-Verdun, D., Hubert, J., and Bouteille, M. Silver staining of NORs in electron microscopy, Exp. Cell Res. 123, 449 (1979). 12. Sommerville, J. Organizing the nucleolus, Nature(London) 318, 410 (1985). 13. Cracker, J., and Nar, P. Nucleolar organizer regions in lymphomas, J. Pathol. 151, 111 (1987). 14. Likovsky, Z., Fabian, E., and Smetana, K. Interphasic nucleolus organizer regions visualized by silver reaction in human hepatocytes, Hepato-gostroenterology 34, 106 (1987).
ONCOLOGY
39, 309-313 (1990)
Nucleolar Organizer Regions in Malignant Transformation of Uterine Cervix YASUHIRO YOKOYAMA,
M.D.,’
DILBAZ
SERDAR, M.D.,
KENNJI NIWA,
M.D.,
AND TERUHIKO
TAMAYA,
M.D.
Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu University, Gifu City, Japan Received March 22, 1990
Nucleolarorganizerregions(NORs) in 70 casesof precancerous and cancerouslesionsof the uterine cervix wereinvestigated.The greater the degreeof dysplasia,the higher the meannumber of silver-stainedNOR (Ag-NOR) dots (mild dysplasia,2.2; moderate dysplasia,2.5; severedysplasia,3.1). The meannumberof dots in moderateor severedysplasiawassignificantly higher than that in normal squamousepithelial cells. The mean numbersof dots in carcinomain situ (CIS) and invasive carcinomawere 3.8 and 3.7, respectively, and weresignificantly higherthan thosein normal squamous epithellalcells,mild dysplasia,moderatedysplasia, and severedysplasia.The meannumber of Ag-NOR dots in the casesof mild dysplasiathat progressedto CIS was 2.7, which wassignlflcantly higher than that in the casesthat regressed.In precancerouslesions,the correlationcoefficientbetweenthe mean number of Ag-NOR dots and the mitotic index was 0.552 (P < O.Ol), whereasno correlation wasnoted betweenthesemeasurementsin CIS and invasive carcinoma.In conclusion,the number of Ag-NOR dots is a good marker to detect cell proliferation in Aeadtic precancerouslesionsand their maliguanttendency. 9 1990 press, Inc.
INTRODUCTION
Nucleolar organizer regions (NORs) are specific sites of genes involved in the formation of the nucleolus in the human being, and are localized in the second constrictions of chromosomes 13, 14, 15, 21, and 22. The NORs can be demonstrated by silver staining, which is thought to visualize the acid proteins surrounding these genes [ 11. Silver-stained NORs (Ag-NORs) observed in the interphase of the cell cycle are ordinarily localized in nucleoli and are considered to contribute to the transcription and/or maturation of rRNA [2]. The intensity of silver staining is thought to have relevance to the I To whom correspondence Gifu City, 500, Japan.
should be sent at Tsukasa-machi
40,
transcriptional activity or potential of such genes [3] and to the progress of the malignant transformation [4,5]. Squamous cell carcinoma of the uterine cervix is speculated to originate in reserve cell proliferation, to which neoplastic initiation might be brought by unknown causes. The present study was designed to investigate quantitative changes in the Ag-NORs of squamous cells in the uterine cervix as a marker of malignant transformation. MATERIALS
AND METHODS
Seventy neoplastic lesions of the uterine cervix comprising 18 cases of mild dysplasia, 12 cases of moderate dysplasia, 17 cases of severe dysplasia, 11 cases of carcinoma in situ (CIS), 12 cases of invasive carcinoma, and 18 cases of normal uterine cervix (9 cases of normal cervical epithelium and 9 cases of squamous metaplasia) were studied. Mild, moderate, and severe dysplasia were defined as follows: mild, neoplastic cells within the lowest third of the epithelium; moderate, into the middle third; and severe, into the upper third. The samples were surgically resected or biopsied specimens obtained in Gifu University Hospital in the past 10 years. They were fixed in 10% neutral formalin and embedded in paraffin. Specimens were selected randomly with the exception of 5 cases of mild dysplasia, 5 cases of moderate dysplasia, and 9 cases of severe dysplasia that had subsequently progressed to carcinoma in situ. Two sequential 3-pm-thick sections were cut and deparaffinized. One section was used for Ag-NOR staining, the other for hematoxylin and eosin (HE) staining. The HE sections were examined to confirm the diagnosis and the mitotic index per 1000 neoplastic cells evaluated. The procedure for Ag-NOR staining was as follows: a solution, made up of 2% gelatin in 1% aqueous formic acid, was mixed with a twofold volume of 50% aqueous
309 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
FIG. 1.
Normal
FIG. 2. Moderate dysplasia. Ag-NOR dots. x530.
epithelium.
The basal and parabasal cells contain distinct and relatively
The dysplastic
cells have distinct Ag-NORs, 310
large Ag-NORs.
x530.
but the mature cells in the upper layer do not contain identifiable
NUCLEOLAR
FIG.
3.
ORGANIZER
REGIONS
IN UTERINE
Carcinoma in situ. The neoplastic cells contain increased numbers of small Ag-NOR dots.
silver nitrate. The mixture was poured on the sections, and incubated at room temperature for 30 min. The staining reaction was terminated to submerge the sections in running tap water. Stained sections were examined under an oil immersion lens at a magnification of 1000, and over 200 nuclei were studied. Cells with nuclear pyknosis were not evaluated because of the unreliability of Ag-NOR staining. Ag-NOR dots in each nucleus were counted, and the
311
CERVIX
X
1060.
mean number of Ag-NOR dots per nucleus was calculated for each section. Data obtained were evaluated by Student’s t test. RESULTS In normal squamous epithelium, the Ag-NORs were clearly observed only in cells of the basal and parabasal layers (Fig. 1); the cells in the superhcial layer did not
312
YOKAYAMA
ET AL.
AgW3?s Precancerous
lesion
(/Cell)
51
-
. +2.07442
i J0 .
0
IO 20 30 40 50 60 70 Mitotic
index
(/lOOOcells)
Carcinoma
Ag-NORs
(/Cell)
61
;I . . , , . ;=,,,,6 0 cases
whose
lesions
have
progressed
to CIS afterward
FIG. 4. Mean numbers of A&NOR dots per nucleus of specimens examined.
show distinct Ag-NOR dots because of pyknosis. The neoplastic cells in moderate dysplasia showed clear and relatively large Ag-NOR dots (Fig. 2), but cells that had matured in the upper layer did not. Carcinoma cells generally had small but increased numbers of Ag-NOR dots (Fig. 3). The mean number of Ag-NOR dots for each diagnostic category is shown in Fig. 4. In normal squamous epithelial cells and metaplastic squamous cells, the mean (&SD) numbers were 2.0 rfr 0.3 and 1.7 2 0.2, respectively. The more advanced the degree of atypia, the greater the mean number of Ag-NOR dots (mild dysplasia, 2.2 + 0.6; moderate dysplasia, 2.5 + 0.4; and severe dysplasia, 3.1 + 0.7). The numbers of Ag-NOR dots in moderate dysplasia (P < 0.01) and severe dysplasia (P < 0.001) were significantly higher than the number in normal squamous epithelial cells. The mean numbers of AgNOR dots in CIS and invasive carcinoma were 3.8 + 0.9 and 3.7 + 0.9, respectively. These numbers were significantly highr than those in normal squamous epithelial cells (P < O.OOl), mild dysplasia (P < O.OOl), moderate dysplasia (P < O.Ol), and severe dysplasia (P < 0.05). However, the difference between the numbers in CIS and invasive carcinoma was not significant. The mean number of Ag-NORs in the cases of mild dysplasia who progressed to ClS was 2.7 + 0.2, which was significantly higher (P < 0.05) than that in the cases that regressed (2.0 ? 0.6) (Fig. 4). As for moderate and
0 10 20 30 40 50 60 70 80 Mitotic index (/lo00 cells)
FIG. 5. Relationship between mitotic index and mean number of Ag-NORs in precancerous lesions and carcinomas.
severe dysplasia, there were no significant differences in the mean numbers of Ag-NOR dots between cases that progressed to CIS (2.6 2 0.3 and 3.2 + 0.6, respectively) and those that did not (2.4 f 0.4 and 3.0 + 0.8, respectively). The relationship between number of Ag-NOR dots and mitotic index in the precancerous lesions and in carcinoma is shown in Fig. 5. The correlation coefficient between these measurements in precancerous lesions was 0.552 (P < O.Ol), whereas no such correlation was recognized in cases of CIS and invasive carcinoma. DISCUSSION The nucleolus is a cellular factory that produces rRNA. Both the number and the size of the nucleoli are related to cellular activity. The genes that are involved in the formation of the nucleolus are localized in specific sites of defined chromosomes as observed during metaphase. With the progression of the cell cycle, the nucleolus can be formed around such genes (rDNA). These genes were first visualized by Goodpasture et al. [ 11 over a decade ago and the methodology subsequently became more stable and reliable [6]. The substance stained by the silver method is not considered to be the rDNA gene itself, but to consist of nonhistone proteins that exist in the vicinity of rDNA
NUCLEOLAR
ORGANIZER
REGIONS IN UTERINE
and play a role in the production and/or maturation of rRNA [7]. Lischwe et al. [8] proved by two-dimensional polyacrylamide gel electrophoresis that the major substances stained by silver in the Novikoff hepatoma ascites cell are proteins B23 and C23. Electron microscopic examination demonstrates that Ag-NOR dots are localized in fibrillar centers and around dense granular components [9-l I]. Cytochemical investigations show that fibrillar centers contain rDNA and numerous proteins (Ag-NOR proteins, RNA polymerase I, and C23 protein) and that dense fibrillar components contain pre-rRNP complexes and Ag-NOR proteins [ 121. The silver stainability may be considered a marker of both rDNA transcription activity and/or transcription potential. Cracker and Nar [ 131 reported that the number of Ag-NORs in high-grade lymphomas was significantly higher than that in low-grade lymphomas. Likovsky et al. [14] reported that the number of Ag-NORs in hepatoma cells was significantly higher than that in normal hepatocytes. Those results suggest that the number of Ag-NORs is related to neoplastic transformation and malignant potential in some species of cells. Uterine cervical squamous cell carcinomas develop in the transformation zone where squamous metaplasia frequently occurs. The metaplasia is hypothesized to result from proliferation and squamous metaplasia of reserve cells. The initiation of the transformation of reserve cells to precancerous lesions or carcinoma remains to be clarified. Of great importance is the discrimination between metaplastic cells and neoplastic cells. With silver staining, the metaplastic cells have a relatively small number of Ag-NOR dots. In contrast, in the intraepithelial neoplastic lesions, the more severe the dysplastic change, the greater the mean number of Ag-NORs dot is. These results are in agreement with those of Rowlands [4] and Egan et al. [5]. However, once the lesion progresses to carcinoma, the mean number of Ag-NORs does not change. In addition, the mean number of Ag-NORs dots in mildly dysplastic lesions that progress to CIS is higher than in those that regress. These results suggest that the number of Ag-NORs dots is related to the malignant potential of intraepithelial neoplasia. In intraepithelial neoplasia, the number of Ag-NOR dots is positively correlated to the mitotic index. How-
CERVIX
313
ever, this is not true in carcinomas. The former finding suggests that the number of Ag-NOR dots is also related to proliferative activity in intraepithelial neoplasia. ACKNOWLEDGMENTS We are very grateful to Associate Professor Shimokawa (Department of Central Laboratory, Gifu University Hospital) and Professor Mori (Department of 1st Pathology, Gifu University School of Medicine) for their contributions to this study.
REFERENCES 1. Goodpasture, C., and Bloom, S. E. Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining, Chromosoma (Berlin) 53, 37 (1975). 2. Carneiro, V., Deltour, R., Fakan, S., et al. The nucleolus and the nucleolar organizer regions, Viol. Cell. 56, 189 (1986). 3. Hofgirtner, F. J., Krone, W., and Jain, K. Correlated inhibition of ribosomal RNA synthesis and silver staining by actinomycin D, Hum. Genet. 47, 329 (1979). 4. Rowlands, D. C. Nucleolar organizer regions in cervical intraepithelial neoplasia, J. Clin. Pathol. 41, 1200 (1988). 5. Egan, M., Freeth, M., and Cracker, J. Relationship between intraepithelial neoplasia of the cervix and the size and number of nucleolar organizer rkgions, Gynecol. Oncol. 36, 30 (1990). 6. Howell, W. M., and Black, D. A. Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: A l-step method, Experientiu 36, 1014 (1980). 7. Hernandez-Verdum, D. (Ed.). The nucleolar organizer regions (collective review), Biol. Cell. 49, I91 (1983). 8. Lischwe, M. A., Smetana, K., Olson, M. 0. J., and Busch, H. Proteins C23 and B23 are the major nucleolar silver staining proteins, Life Sci. 25, 701 (1979). 9. Schwarzacher, H. G., Mikelsaar, A. V., and Schnedl, W. The nature of the nucleolus organizer regions: Electron- and light-microscopic studies on human cells in interphase, mitosis, and meiosis, Cytogenet. Cell Genet. 20, 24 (1978). 10. Goessens, G. Localisation of nucleolus-organising regions in interphase cells, Cell Tissue Res. 200, 159 (1979). Il. Bourgeois, C. A., Hernandez-Verdun, D., Hubert, J., and Bouteille, M. Silver staining of NORs in electron microscopy, Exp. Cell Res. 123, 449 (1979). 12. Sommerville, J. Organizing the nucleolus, Nature(London) 318, 410 (1985). 13. Cracker, J., and Nar, P. Nucleolar organizer regions in lymphomas, J. Pathol. 151, 111 (1987). 14. Likovsky, Z., Fabian, E., and Smetana, K. Interphasic nucleolus organizer regions visualized by silver reaction in human hepatocytes, Hepato-gostroenterology 34, 106 (1987).
