A rapid method to determine proliferation patterns of normal and ...

American Journal ofPathology, Vol. 136, No. 4, April 1990 Copyright © American Association ofPathologists

Rapid Communication A Rapid Method to Determine Proliferation Patterns of Normal and Malignant Tissues by H3 mRNA In Situ Hybridization

Ming Yung Chou,* Allen L. C. Chang,t Jim McBride,t Bruce Donoff,§ George T. Gallagher,t and David T. W. Wongt From the Departments of Oral Medicine and Oral

Pathologyt and Oral and Maxillofacial Surgery,§ Harvard School ofDental Medicine, Boston, Massachusetts; and Chung Shan Medical and Dental College, Taichung, Taiwan, Republic ofChina*

A general method applicable for the determination of any mammalian tissue's proliferative pattern is described. This method determines the cellular mRNA level of a proliferation-dependent gene, histone H3, by in situ hybridization. The cell-cycle Sphase-specific expression of this highly conserved ubiquitous cellular gene, and the lack of it in resting cells, permits the unambiguous identification of cycling cells in any tissues, normal or diseased. This method can be conveniently coupled with routine biopsy and could be streamlined for a central laboratory with results obtainable in 2 days. Furthermore, this procedure works successfully on formalin-fixed paraffin-embedded sections, thus allowing retrospective studies of biopsies or autopsy materials. (Am JPathol 1990, 136:729-733)

One of the major defects in cancer is the deregulated growth of the malignant tissue. It is believed generally that the higher the proliferation rate, the more aggressive is the tumor, and subsequently the prognosis is less favorable. Treatment modalities of cancers, namely radiation, chemotherapy, and surgery, depend largely on the proliferative index of the tumor. Thus determining the proliferative index of tumors is of primary importance in the diagnosis of cancer and in subsequent treatment decisions. At present, measurement of tumor proliferative index is ac-

complished by morphologic quantitation of mitotic index, thymidine-labeling index, and flow cytometry.1-3 Most recently, Hoffman et a14 have demonstrated an elegant technique for the in vitro determination of the proliferative capacity of normal and malignant human tissues. Each of these methods has its own merits and limitations. Most require in vitro manipulation of the tumor tissue. Examination of mitotic figures is the most common and most physiologic approach; however, it is time consuming and requires an experienced pathologist. Furthermore, the mitotic phase of the cell cycle is only about one fifth of the cell cycle time; most cycling cells not in the mitotic phase are not detectable. We describe here a simple method for determining the proliferative pattern of any tissue, normal or diseased. It is theoretically applicable to any mammalian species. The application uses the advantage of the in situ hybridization technique, which allows the simultaneous examination of histologic features and detection of cellular mRNA levels. The expression of the histone H3 gene is known to be tightly coupled to DNA synthesis in all cell types examined (normal and malignant).5 In cycling cells, the level of H3 mRNA reaches a peak at the S phase and then rapidly disappears toward the end of G2 . Cells traversing through the mitotic (M) phase will contain little or no H3 mRNA. Histone mRNAs are not polyadenylated and are rapidly degraded when the S phase is completed. Nondividing cells will contain no H3 mRNA. Cycling cells traversing G1, S, or G2 phases of the cell cycle will contain detectable levels of H3 mRNA (about four fifths of the cell cycle time). Thus examining the cellular levels of H3 Supported by Public Health Service grant DE-08680 and in part by grant 0226 from the Smokeless Tobacco Research Council, Inc. David T. W. Wong is supported by a Cancer Research Scholar Award from the American Cancer Society, Massachusetts Division. George T. Gallagher is supported by a Milton Fund Grant from the Harvard Medical School. Address reprint requests to David T. W. Wong, Department of Oral Medicine and Oral Pathology, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115.

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mRNA by in situ hybridization will allow us to identify most of the proliferating cells. Theoretically, if coupled to mitotic index examination, all dividing cells can be identified.

the probe used for in situ hybridization is about 2 X 106 cpm per section.

In Situ Hybridization

Materials and Methods

Cell Culture OEC-M1 s are human oral epidermoid carcinoma cells derived from a gingival carcinoma.6 They are maintained at 370C, 5% C02 in DMEM medium supplemented with 10% fetal bovine serum and antibiotics (penicillin, 100 units per ml; streptomycin, 100 Ag per ml; and amphotericin B, 0.25 jig per ml; Whittaker MA Bioproducts, Walkersville, MD). For [3H]-thymidine labeling, cells in S phase were labeled with [3H]-thymidine (Amersham, Arlington Heights, IL; 16.0 Ci/mmol), at 1.0 gCi/ml of medium for 15 minutes before fixation. For DNA synthesis inhibition experiments, 10 mmol of hydroxyurea (Sigma Chemical Co, St Louis, MO, H-8627) was administered to exponentially growing OEC-Ml cells for 1 hour.

Human Oral Cancers Freshly resected human oral epidermoid carcinomas were obtained from the Department of Oral and Maxillofacial Surgery at the Massachusetts General Hospital. They were immediately fixed in freshly prepared 4% paraformaldehyde and processed for in situ hybridization according to the protocol of Zeller et al.7 Previously formalinfixed human oral cancers were obtained from the Department of Oral Pathology at the Harvard School of Dental Medicine.

Molecular Probe The rat histone H3 cDNA probe was provided to us by Dr. William F. Marzluff.8 This cDNA was recloned into the plasmid vector pGEM3 (Promega, Madison WI), such that antisense transcripts will be generated by the T7 promoter. Production of labeled antisense H3 riboprobe was done by using a RNA transcription kit (Promega, Madison WI), 35S-UTP (Amersham, SJ.40383, SP6/T7 Grade, 850Ci/mol), and T7 RNA polymerase (Stratagene, La Jolla, CA). Typically, 80% to 90% incorporation was obtained. Each in vitro transcription reaction yields about 230 ng of synthesized RNA with a specific activity of about 3 X 108 cpm//ug RNA. The final specific activity of

In situ hybridization was done according to the method of Zeller et al,7 with minor modifications.9

Results In vitro cultures of human epidermoid carcinoma cell lines were used to determine if intracellular levels of the histone H3 mRNA can be used to detect proliferating cells by in situ hybridization, using an approach similar to that employed by Lawrence et al.'0 OEC-M1 is a human oral carcinoma cell line derived from a gingival epidermoid carcinoma.6 Exponentially growing OEC-M1 cells were fixed and processed for in situ hybridization with an 3S-labeled H3 antisense(-)riboprobe8 (Figure 1 A). Subsequent autoradiography revealed a bimodal population of cells in which approximately 47% of cells contained grain densities that were significantly higher than that of background. Consistent with the known cell-cycle expression pattern of H3, tumor cells in the mitotic phase contain little or no detectable H3 mRNA (Figure 1 A, arrows). Parallel cultures of OEC-M1 cells labeled with [3H]-thymidine followed by autoradiography revealed that approximately 45% were found to be in S phase. Thus a good agreement was obtained between metabolic labeling by [3H]-thymidine- and H3 mRNA-positive cells. Both the incorporation of [3H]thymidine into the nuclei and H3 mRNA labeling by in situ hybridization were eliminated by an incubation of these cultures with the DNA synthesis inhibitor hydroxyurea at 10 mmol for 1 hour before fixation, confirming that the expression of H3 is linked to active DNA synthesis.5 Control hybridization with a 35S-labeled mouse EGF RNA probe did not result in any labeling of these tumor cells (data not shown). These results indicate that H3 mRNA labeling of human oral tumor cells by in situ hybridization is a valid procedure to mark the population of dividing cells. These in vitro results led us to entertain the possibility of using this approach to determine the proliferative pattern of normal and diseased tissues. Clinically resected normal and malignant oral tissues were processed for in situ hybridization as described by Zeller et al,7 and subjected to in situ hybridization examination for H3 mRNA using the antisense H3 riboprobe followed by autoradiography. Figure 1 B is a section of normal gingival tissue hybridized with the 35S-labeled antisense H3 riboprobe. Despite the tangential nature of the section, only an occa-

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Figure 1. In situ bybridization detection of histone H3 mRVA in normal and malignant human oral epithelium. Preparation of35SUTP-labeled antisense mouse H3probe and the protocol ofin situ hybridization is according to the method of Zeller et al. 72.5 X 105 cpm of the probe was usedfor each section in a hybridization mixture containing 50%formamide. Hybridization is for 18 hours at 50' C. All sections were counterstained with Giemsa (Fisher Scientiflc). A: An island of OEC-Ml cells grown on a glass side. Arrows point to mitotic figures. Exposure time is 48 hours at 4 C Original magnification: 200X. B: Normal gingival epithelium (tangential cut). Original magniication, 31X. C: A well-differentiated human oral epidermoid carcinomafreshlypreparedforin situ hybridization according to the method of Zeller et al.' Original magnification, 80X. D: A moderately dif[erentiated human oral epidermoid carcinoma. Original magnification, 80U. E: A high-power view of an invasive island of the tumor in D. Arrow points to a mitotic figure. Original magnification, 20(X. F: A well-differentiated human oral epidermoid carcinoma that was initiallyfixed informalin and embedded in paraffinfor 10years. Original magnification, 801X. Exposure timefor sections in B to F was 1 week at 4' C

sional basal cell is labeled. This corresponds with the known fact that in normal epithelium the proliferative compartment resides primarily in the basal layer.1" Figures 1C and D show two different human oral carcinomas exhibiting features of well and moderate differentiation, respec-

tively. The central tumor island in the well-differentiated oral tumor (Figure 1 C) displays a H3 labeling pattern that is predominantly toward the periphery. The deeply invasive carcinoma in panel D, on the other hand, displays an H3 labeling pattern that suggests that most cells in the

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invading strands and cords of the malignant epithelium are actively dividing. Notice that an occasional cell in the stromal portion of these tumors also scored positive. These are likely to be of inflammatory or endothelial origin. Figure 1 E demonstrates an internal control of the hybridization with the H3 probe. A mitotic figure from the tumor in panel 1 E can be seen (arrow) to be practically devoid of H3 labeling, while the surrounding tumor cells display a heterogeneous pattern of H3 labeling. Next we tried to determine if we can perform similar studies on tumor tissues collected from previous biopsies that were initially fixed in formalin and embedded in paraffin. Figure 1 F is a biopsy specimen from a human oral cancer, formalin fixed and embedded in paraffin 10 years earlier. This well-differentiated epidermoid carcinoma labeled very prominently with our 35S-labeled antisense H3 RNA probe, clearly demonstrating dysplasia in the surface epithelium (to the left) and rapid cellular division in the invading tumor islands. We examined 10 different human oral cancers, each block at least 10 years old, and found all to label satisfactorily. Control hybridization of serial sections to the tissues shown in 1 B to F with a 35Slabeled mouse EGF antisense RNA probe did not label any cells.

Discussion In this report we have shown the successful demonstration of the cellular H3 mRNA by in situ hybridization in normal and malignant epithelial tissues and its usefulness as an indicator of dividing cells. Similar to the results obtained by Lawrence et al10 using human WI-38 fibroblasts, labeling of human oral tumor cells in situ for H3 mRNA marked dividing cells. The similar percentage of H3 mRNA-positive cells to [3H]-thymidine metabolic labeling, abolishment of H3 mRNA labeling by hydroxyurea treatment, and no labeling of cells in the mitotic phase all indicate that the in situ H3 mRNA labeling is specific and can mark dividing cells in vitro. Next we demonstrated the in situ labeling of H3 mRNA in human normal and malignant oral epithelium. From the pattern of H3 mRNA labeling in normal and malignant tissues and the lack of H3 mRNA labeling in identifiable mitotic figures allowed us to conclude that in situ H3 mRNA labeling can be used to mark dividing cells in normal and malignant human oral epithelium. Because of the highly conserved nature of the H3 gene and its ubiquitous expression in eukaryotic cells, this application should be useful in any mammalian species whenever a modification of growth pattern needs to be determined. Compared to the other existing methods for determining cellular proliferation, this application has the

following advantages. First, this technique can be conveniently coupled with routine biopsy and thus could be considered noninvasive. Second, normal or diseased tissues can be examined. Third, minimal tissue is required. Fourth, the tissue to be examined is in its native state with regard to tissue architecture. Fifth, the proliferative status of a tumor at the time of biopsy is determined, instead of a cumulative index over an extended period of time. Sixth, this technique works successfully on formalin-fixed, paraffin-embedded sections, thus allowing retrospective studies of previously collected tissues. Last, this technique can be streamlined and coupled to a nonisotopic detection procedure, with results obtainable on frozen sections in as quickly as 2 working days. The only drawback of this technique is that we determine the proliferation status at a single point in time. However, it is not clear whether a 3week in vitro manipulation of the tumor tissue involving [3H]-thymidine incorporation for a 4-hour labeling period is better than a cross-sectional determination of a tumor's proliferation capacity. One could argue that the singlepoint determination of a tumor's proliferative index is more valuable when speed and information regarding a neoplastic lesion's proliferative index is important in determining subsequent treatment modalities. This simple application provides a convenient genetic marker for cellular proliferation that should serve well not only for determining tumor proliferative capacity on routine histopathologic sections but also for applications in experimental pathology in which an alteration of proliferation capacity must be assayed without previous administration of a tracer substance.

References 1. Meyer JS, Conner RE: In vitro labelling of solid tissues with tritiated thymidine for autoradiographic detection of S-phase nuclei. Stain Tech 1977, 52:185-195 2. McDivitt R, Stone K, Meyer J: A method for dissociation of viable human breast cancer cells that produces flow cytometric kinetic information similar to that obtained by thymidine labelling. Cancer Res 1984, 44:2628-2633 3. McDivitt RW, Stone KR, Craig RB, Palmer JO, Meyer J, Bauer WC: A proposed classification of breast cancer based on kinetic information. Cancer 1986, 57:269-276 4. Hoffman Robert M, Conners Kenneth M, Meerson-Monosov Ann Z, Herrera Hector, Price Jeffrey H: A general native-state method for determination of proliferation capacity of human normal and tumor tissues in vitro. Proc Natl Acad Sci USA

1989, 86:2013-2017 5. Stein GS, Stein JL, Marzluff WF: Histone Genes. Structure, Organization and Regulation. New York, Wiley, 1984. 6. Meng CL, Tu C-L, Chang L-C, Hsu S-F, Wu CH: Molecular Biology of Neoplasia. Edited by EH Chang, JK Lin, PC Huang. Taipei, Republic of China, Academia Sinica, 1985

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7. Zeller R, Bloch KD, Williams BS, Arceci RJ, Seidman CE: Localized expression of the atnal natriuretic factor gene durng embryogenesis. Gene and Development 1987,1:693-698 8. Sittman DB, Chiu I-M, Pan C-J, Cohn RH, Kedes LH, Marzluff WF: Isolation of two clusters of mouse histone genes. Proc Natl Acad Sci USA 1981, 78:4078-4082 9. Chang LC, Chou MY, Chow P, Matossian K, McBride J, Chiang T, Gallagher G, Wong DTW: Detection of TGF-a

mRNA in normal and chemically transformed hamster oral epithelium. Cancer Research 1989, 49:6700-6707 10. Lawrence HB, Singer RH, Villnave CA, Stein JL, Stein GS: Intracellular distribution of histone mRNA in human fibroblasts studied by in situ hybridization. Proc Natl Acad Sci

USA 1988, 85:463-467 11. Schroeder HE: Differentiation of human oral stratified epithelium. New York, S. Kruger, 1981.