Papillary Thyroid Carcinoma Oncogene (RET/PTC) Alters the Nuclear Envelope and Chromatin Structure
1998; Elsevier BV; Volume: 153; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65731-8
ISSN1525-2191
AutoresAndrew H. Fischer, Jane Bond, Panya Taysavang, O. Eugene Battles, David Wynford‐Thomas,
Tópico(s)Nuclear Structure and Function
ResumoCurrent evidence suggests the papillary thyroid carcinoma oncogene (RET/PTC) generates papillary thyroid carcinomas in one genetic step. We tested a resulting prediction that RET/PTC expression in thyroid epithelium should be sufficient to cause the changes in nuclear morphology diagnostic of this tumor. Primary cultures of human thyroid epithelial cells were infected with a RET/PTC retroviral construct. Morphological scoring by two independent cytopathologists shows RET/PTC expression by immunohistochemistry to be highly associated (p ≪ 0.0001) with an irregular nuclear contour and a euchromatic appearance compared with non-expressing cells in the same cultures. The altered nuclear morphology is not due to gene transfer or transformation per se as primary thyroid cell cultures infected with a retroviral H-RAS construct differ from RET/PTC-infected cells by showing round nuclear envelopes and coarser chromatin, as determined by the independent scoring of two cytopathologists (p ≪ 0.0001). In addition, RET/PTC-transfected cells appear to disperse, whereas RAS-transfected cells grow as discrete colonies. The results provide additional support for the hypothesis that RET/PTC is sufficient to cause papillary thyroid carcinomas. A signaling pathway downstream of RET/PTC leads to restructuring of the nuclear envelope and chromatin, and the signal does not depend entirely, if at all, on a RAS pathway. Current evidence suggests the papillary thyroid carcinoma oncogene (RET/PTC) generates papillary thyroid carcinomas in one genetic step. We tested a resulting prediction that RET/PTC expression in thyroid epithelium should be sufficient to cause the changes in nuclear morphology diagnostic of this tumor. Primary cultures of human thyroid epithelial cells were infected with a RET/PTC retroviral construct. Morphological scoring by two independent cytopathologists shows RET/PTC expression by immunohistochemistry to be highly associated (p ≪ 0.0001) with an irregular nuclear contour and a euchromatic appearance compared with non-expressing cells in the same cultures. The altered nuclear morphology is not due to gene transfer or transformation per se as primary thyroid cell cultures infected with a retroviral H-RAS construct differ from RET/PTC-infected cells by showing round nuclear envelopes and coarser chromatin, as determined by the independent scoring of two cytopathologists (p ≪ 0.0001). In addition, RET/PTC-transfected cells appear to disperse, whereas RAS-transfected cells grow as discrete colonies. The results provide additional support for the hypothesis that RET/PTC is sufficient to cause papillary thyroid carcinomas. A signaling pathway downstream of RET/PTC leads to restructuring of the nuclear envelope and chromatin, and the signal does not depend entirely, if at all, on a RAS pathway. Thyroid epithelial cells can be transformed via two different pathways, a follicular pathway and a papillary pathway. The two types of resulting thyroid epithelial neoplasms are distinctive in their clinical features and morphology1LiVolsi VA Surgical Pathology of the Thyroid.in: Bennington JL series Major Problems in Pathology. vol. 22. WB Saunders, Philadelphia1990Google Scholar, 2Rosai J Carcangiu ML Delellis RA Tumors of the Thyroid Gland, Fascicle 5, third series.in: Rosai J Sobin LH Armed Forces Institute of Pathology, Washington, DC1992Google Scholar, 3Franssila KO Ackerman LV Brown CL Hedinger CE Follicular carcinoma.Semin Diagn Pathol. 1985; 2: 101-122PubMed Google Scholar as well as their genetics.4Lemoine NR Mayall ES Wyllie FS Williams ED Goyns M Stringer B Wynford-Thomas D High frequency of ras oncogene activation in all stages of human thyroid tumorigenesis.Oncogene. 1989; 4: 159-164PubMed Google Scholar, 5Wright PA Lemoine NR Mayall ES Wyllie FS Hughes D Williams ED Wynford-Thomas D Papillary and follicular thyroid carcinomas show a different pattern of ras oncogene mutation.Br J Cancer. 1989; 60: 576-577Crossref PubMed Scopus (84) Google Scholar, 6Santoro M Carlomagno F Hay ID Herrmann MA Grieco M Melillo R Pierotti MA Bongarzone I Della Porta G Berger N Peix JL Paulin C Fabien N Vecchio G Jenkins RB Fusco A Ret oncogene activation in human thyroid neoplasms is restricted to the papillary cancer subtype.J Clin Invest. 1992; 89: 1517-1522Crossref PubMed Scopus (341) Google Scholar, 7Santoro M Grieco M Melillo RM Fusco A Vecchio G Molecular defects in thyroid carcinomas: role of the RET oncogene in thyroid neoplastic transformation.Eur J Endocrinol. 1995; 133: 513-522Crossref PubMed Scopus (66) Google Scholar, 8Wynford-Thomas D Origin and progression of thyroid epithelial tumours: cellular and molecular mechanisms.Hormone Res. 1997; 47: 145-157Crossref PubMed Scopus (119) Google Scholar RAS mutations are common in the follicular pathway and appear to be an early event.4Lemoine NR Mayall ES Wyllie FS Williams ED Goyns M Stringer B Wynford-Thomas D High frequency of ras oncogene activation in all stages of human thyroid tumorigenesis.Oncogene. 1989; 4: 159-164PubMed Google Scholar, 8Wynford-Thomas D Origin and progression of thyroid epithelial tumours: cellular and molecular mechanisms.Hormone Res. 1997; 47: 145-157Crossref PubMed Scopus (119) Google Scholar It seems likely that follicular carcinomas arise from follicular adenomas, although there is no direct supporting evidence. The defining feature of malignancy in the follicular pathway is vascular or capsular invasion.1LiVolsi VA Surgical Pathology of the Thyroid.in: Bennington JL series Major Problems in Pathology. vol. 22. WB Saunders, Philadelphia1990Google Scholar, 2Rosai J Carcangiu ML Delellis RA Tumors of the Thyroid Gland, Fascicle 5, third series.in: Rosai J Sobin LH Armed Forces Institute of Pathology, Washington, DC1992Google Scholar, 3Franssila KO Ackerman LV Brown CL Hedinger CE Follicular carcinoma.Semin Diagn Pathol. 1985; 2: 101-122PubMed Google Scholar Nuclear morphological features overlap considerably between follicular carcinomas, follicular adenomas, and even normal thyroid epithelium.3Franssila KO Ackerman LV Brown CL Hedinger CE Follicular carcinoma.Semin Diagn Pathol. 1985; 2: 101-122PubMed Google Scholar, 9Johannessen JV Sobrinho-Simoes M Well differentiated thyroid tumors: problems in diagnosis and understanding.Pathol Annu. 1983; 18: 255-285PubMed Google Scholar, 10Salmon I Kiss R Franc B Gasperin P Heimann R Pasteels JL Verhest A Comparison of morphonuclear features in normal, benign and neoplastic thyroid tissue by digital cell image analysis.Anal Quant Cytol Histol. 1992; 14: 47-54PubMed Google Scholar The papillary pathway is defined by a combination of morphological features, the most important of which are nuclear alterations: nuclear contour irregularities, such as nuclear grooves and inclusions, and dispersal of heterochromatin aggregates into a finely textured (ground-glass) chromatin.1LiVolsi VA Surgical Pathology of the Thyroid.in: Bennington JL series Major Problems in Pathology. vol. 22. WB Saunders, Philadelphia1990Google Scholar, 3Franssila KO Ackerman LV Brown CL Hedinger CE Follicular carcinoma.Semin Diagn Pathol. 1985; 2: 101-122PubMed Google Scholar, 11Vickery Jr, AL Carcangiu ML Johannessen JV Sobrinho-Simoes M Papillary carcinoma.Semin Diagn Pathol. 1985; 2: 90-100PubMed Google Scholar Unlike most other solid tumors, including follicular thyroid carcinoma, there is no recognized benign counterpart of papillary thyroid carcinoma. Rather, papillary thyroid carcinomas appear histologically to arise in a single step; the unusual nuclear morphology diagnostic of this tumor can be identified within occult, relatively homogeneous foci composed of very few cells,12Hay ID Grant CS van Heerden JA Goellner JR Ebersold JR Bergstralh EJ Papillary thyroid microcarcinoma: a study of 535 cases observed in a 50-year period.Surgery. 1992; 112: 1139-1147PubMed Google Scholar and such occult tumors do not appear to arise from a morphologically altered population. The papillary thyroid carcinoma oncogene (RET/PTC) is a rearranged version of the tyrosine kinase RET, the receptor for glial-cell-line-derived neurotrophic factor.13Jing S Wen D Yu Y Holst PL Luo Y Fang M Tamir R Antonio L Hu Z Cupples R Louis JC Hu S Altrock BW Fox GM GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-α, a novel receptor for GDNF.Cell. 1996; 85: 1113-1124Abstract Full Text Full Text PDF PubMed Scopus (1058) Google Scholar RET/PTC rearrangement is nearly restricted to papillary thyroid carcinomas. No rearrangement of RET/PTC has ever been detected in more than 500 examples of nonthyroid tumors,14Santoro M Sabino N Ishizaka Y Ushijima T Carlomagno F Cerrato A Grieco M Battaglia C Martelli ML Paulin C Fabien N Sugimura T Fusco A Nagao M Involvement of RET oncogene in human tumours: specificity of RET activation to thyroid tumours.Br J Cancer. 1993; 68: 460-464Crossref PubMed Scopus (74) Google Scholar and RET/PTC rearrangements in nonpapillary thyroid lesions are restricted to a very small percentage of possible follicular-type lesions.6Santoro M Carlomagno F Hay ID Herrmann MA Grieco M Melillo R Pierotti MA Bongarzone I Della Porta G Berger N Peix JL Paulin C Fabien N Vecchio G Jenkins RB Fusco A Ret oncogene activation in human thyroid neoplasms is restricted to the papillary cancer subtype.J Clin Invest. 1992; 89: 1517-1522Crossref PubMed Scopus (341) Google Scholar, 15Ishizaka Y Kobayashi S Ushijima T Hirohashi S Sugimura T Nagao M Detection of retTPC/PTC transcripts in thyroid adenomas and adenomatous goiter by an RT-PCR method.Oncogene. 1991; 6: 1667-1672PubMed Google Scholar, 16Bounacer A Wicker R Caillou B Cailleux AF Sarasin A Schlumberger M Suarez HG High prevalence of activating ret proto-oncogene rearrangements in thyroid tumors from patients who had received external radiation.Oncogene. 1997; 15: 1263-1273Crossref PubMed Scopus (274) Google Scholar RET/PTC is activated when RET undergoes a translocation fusing its tyrosine kinase domain to a variety of other 5′ elements, thereby removing the promoter, the extracellular ligand-binding domain, and the membrane-anchoring domain of RET (reviewed in 7Santoro M Grieco M Melillo RM Fusco A Vecchio G Molecular defects in thyroid carcinomas: role of the RET oncogene in thyroid neoplastic transformation.Eur J Endocrinol. 1995; 133: 513-522Crossref PubMed Scopus (66) Google Scholar, 17Jhiang SM Mazzaferri EL The ret/PTC oncogene in papillary thyroid carcinoma.J Lab Clin Med. 1994; 123: 331-337PubMed Google Scholar). RET is expressed in neural-crest-derived tissues, including thyroid C cells, but thyroid follicular cells express at most minimal amounts of RET mRNA.18Jhiang SM Smanik PA Mazzaferri EL Development of a single-step duplex RT-PCR detecting different forms of ret activation and identification of the third form of in vivo ret activation in human papillary thyroid carcinoma.Cancer Lett. 1994; 78: 69-76Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 19Viglietto G Chiappetta G Martinez-Tello FJ Fukunaga FH Tallini G Rigopoulou D Visconti R Mastro A Santoro M Fusco A RET/PTC oncogene activation is an early event in thyroid carcinogenesis.Oncogene. 1995; 11: 1207-1210PubMed Google Scholar In contrast, the 5′ sequences that donate their promoters and become fused to RET are all ubiquitously expressed, including in thyroid follicular epithelium. Three functional consequences of the translocations giving rise to RET/PTC could be significant for its transforming activity: 1) transcriptional activation driven by the new 5′ promoter, 2) oligomerization of RET, through motifs in the 5′ sequences, with resulting constitutive phosphorylation activity,20Tong Q Xing S Jhiang SM Leucine zipper-mediated dimerization is essential for the PTC1 oncogenic activity.J Biol Chem. 1997; 272: 9043-9047Crossref PubMed Scopus (72) Google Scholar, 21Durick K Yao VJ Borrello MG Bongarzone I Pierotti MA Taylor SS Tyrosines outside the kinase core and dimerization are required for the mitogenic activity of RET/ptc2.J Biol Chem. 1995; 270: 24642-24645Crossref PubMed Scopus (38) Google Scholar, 22Klugbauer S Demidchik EP Lengfelder E Rabes HM Detection of a novel type of RET rearrangement (PTC5) in thyroid carcinomas after Chernobyl and analysis of the involved RET-fused gene RFG5.Cancer Res. 1998; 58: 198-203PubMed Google Scholar and 3) loss of membrane association due to deletion of the transmembrane-anchoring sequences of RET in the translocation.7Santoro M Grieco M Melillo RM Fusco A Vecchio G Molecular defects in thyroid carcinomas: role of the RET oncogene in thyroid neoplastic transformation.Eur J Endocrinol. 1995; 133: 513-522Crossref PubMed Scopus (66) Google Scholar, 23Ishizaka Y Shima H Sugimura T Nagao M Detection of phosphorylated retTPC oncogene product in cytoplasm.Oncogene. 1992; 7: 1441-1444PubMed Google Scholar RET/PTC is active in 25% to 80% of papillary thyroid carcinomas.16Bounacer A Wicker R Caillou B Cailleux AF Sarasin A Schlumberger M Suarez HG High prevalence of activating ret proto-oncogene rearrangements in thyroid tumors from patients who had received external radiation.Oncogene. 1997; 15: 1263-1273Crossref PubMed Scopus (274) Google Scholar The incidence of RET/PTC rearrangements is highest in radiation-associated papillary thyroid carcinomas.16Bounacer A Wicker R Caillou B Cailleux AF Sarasin A Schlumberger M Suarez HG High prevalence of activating ret proto-oncogene rearrangements in thyroid tumors from patients who had received external radiation.Oncogene. 1997; 15: 1263-1273Crossref PubMed Scopus (274) Google Scholar, 24Ito T Seyama T Iwamoto KS Mizuno T Tronko ND Komissarenko IV Cherstovoy ED Satow Y Takeichi N Dohi K Akiyama M Activated RET oncogene in thyroid cancers of children from areas contaminated by Chernobyl accident.Lancet. 1994; 344: 259PubMed Google Scholar, 25Klugbauer S Lengfelder E Demidchik EP Rabes HM High prevalence of RET rearrangement in thyroid tumors of children from Belarus after the Chernobyl reactor accident.Oncogene. 1995; 11: 2459-2467PubMed Google Scholar Papillary thyroid carcinomas expressing RET/PTC tend to be small and show classic morphology.26Tallini G Santoro M Helie M Carlomagno F Salvatore G Chiappetta G Carcangiu ML Fusco A RET/PTC oncogene activation defines a subset of papillary thyroid carcinomas lacking evidence of progression to poorly differentiated or undifferentiated tumor phenotypes.Clin Cancer Res. 1998; 4: 287-294PubMed Google Scholar Genetic studies suggest that RET/PTC activation is an early genetic event. In keeping with the histological evidence that papillary thyroid carcinomas appear to arise in one genetic step, five pieces of evidence suggest that RET/PTC per se may be sufficient to mediate the transformation. 1) Occult papillary thyroid carcinomas, composed of very few cells, have been shown to sometimes express RET/PTC.19Viglietto G Chiappetta G Martinez-Tello FJ Fukunaga FH Tallini G Rigopoulou D Visconti R Mastro A Santoro M Fusco A RET/PTC oncogene activation is an early event in thyroid carcinogenesis.Oncogene. 1995; 11: 1207-1210PubMed Google Scholar 2) Immunohistochemistry of RET/PTC-bearing tumors has shown the presence of RET/PTC in all of the tumor cells.22Klugbauer S Demidchik EP Lengfelder E Rabes HM Detection of a novel type of RET rearrangement (PTC5) in thyroid carcinomas after Chernobyl and analysis of the involved RET-fused gene RFG5.Cancer Res. 1998; 58: 198-203PubMed Google Scholar 3) Cytogenetic abnormalities in papillary thyroid carcinomas are sometimes restricted to single translocations involving 10q11.2,27Roque L Clode AL Gomes P Rosa-Santos J Soares J Castedo S Cytogenetic findings in 31 papillary thyroid carcinomas.Genes Chromosomes & Cancer. 1995; 13: 157-162Crossref PubMed Scopus (28) Google Scholar, 28Sozzi G Bongarzone I Miozzo M Cariani CT Mondellini P Calderone C Pilotti S Pierotti MA Della Porta G Cytogenetic and molecular genetic characterization of papillary thyroid carcinomas.Genes Chromosomes & Cancer. 1992; 5: 212-218Crossref PubMed Scopus (49) Google Scholar, 29Sozzi G Bongarzone I Miozzo M Borrello MG Blutti MG Pilotti S Della Porta G Pierotti MA A t(10;17) translocation creates the RET/PTC2 chimeric transforming sequence in papillary thyroid carcinoma.Genes Chromosomes & Cancer. 1994; 9: 244-250Crossref PubMed Scopus (81) Google Scholar the location of RET, suggesting RET/PTC could be the sole abnormality in some tumors. 4) Two transgenic mouse models of RET/PTC, driven by a thyroglobulin promoter, both develop multifocal thyroid tumors at an early age, and the tumors are histologically very similar to human papillary thyroid carcinomas, with "ground glass nuclei, grooves and inclusions".30Jhiang SM Sagartz JE Tong Q Parker-Thornburg J Capen CC Cho JY Xing S Ledent C Targeted expression of the ret/PTC1 oncogene induces papillary thyroid carcinomas.Endocrinology. 1996; 137: 375-378Crossref PubMed Scopus (242) Google Scholar, 31Santoro M Chiappetta G Cerrato A Salvatore D Zhang L Manzo G Picone A Portella G Santelli G Vecchio G Fusco A Development of thyroid papillary carcinomas secondary to tissue-specific expression of the RET/PTC1 oncogene in transgenic mice.Oncogene. 1996; 12: 1821-1826PubMed Google Scholar 5) The most direct evidence that RET/PTC is sufficient to transform thyroid epithelial cells comes from gene transfer studies. When expressed in primary cultures of normal human thyroid epithelium, RET/PTC is able by itself to drive proliferation sufficiently to produce colonies of up to 106 cells with a characteristic fenestrated appearance by phase contrast microscopy.32Bond JA Wyllie FS Rowson J Radulescu A Wynford-Thomas D In vitro reconstruction of tumour initiation in a human epithelium.Oncogene. 1994; 9: 281-290PubMed Google Scholar Following on this observation, the aim of the present study was to determine whether RET/PTC is sufficient to cause the intriguing nuclear morphological alterations characteristic of papillary thyroid carcinoma. Primary cultures of normal human thyroid cells were produced and gene transfer carried out as previously described.32Bond JA Wyllie FS Rowson J Radulescu A Wynford-Thomas D In vitro reconstruction of tumour initiation in a human epithelium.Oncogene. 1994; 9: 281-290PubMed Google Scholar This culture system is ∼99% epithelial as judged by cytokeratin immunostaining.32Bond JA Wyllie FS Rowson J Radulescu A Wynford-Thomas D In vitro reconstruction of tumour initiation in a human epithelium.Oncogene. 1994; 9: 281-290PubMed Google Scholar Briefly, 2 days after plating, cells were infected with an amphotrophic (psi-CRIP) RET/PTC-1 retroviral vector, also conferring G418 (neomycin) resistance.32Bond JA Wyllie FS Rowson J Radulescu A Wynford-Thomas D In vitro reconstruction of tumour initiation in a human epithelium.Oncogene. 1994; 9: 281-290PubMed Google Scholar RET/PTC-1 is a naturally occurring papillary thyroid carcinoma oncogene formed from part of a protein called H4, bearing a leucine zipper dimerization domain, fused amino-terminal to the tyrosine kinase domain of RET. Control infections were performed with an activated H-RAS-neomycin retroviral vector (psi-CRIP-DOEJ), as previously described.32Bond JA Wyllie FS Rowson J Radulescu A Wynford-Thomas D In vitro reconstruction of tumour initiation in a human epithelium.Oncogene. 1994; 9: 281-290PubMed Google Scholar Four days after infection, cells were passaged onto Permanox (Nunc, Naperville, IL) plastic tissue culture slides either with or without G418 selection. After one or two more weeks in culture, cells were fixed by immersion in 95% ethanol and then spayed with a solution of polyethylene glycol in alcohol (Fix-Rite, Richard-Allen, Kalamazoo, MI) and stored dry. Modified Papanicolaou staining (Richard-Allen) was performed according to standard protocols except that slides were coverslipped with glycerol (Sigma Chemical Co., St. Louis, MO) instead of xylene-based media after the last absolute alcohol wash, as the slides are plastic. Immunohistochemistry with a polyclonal rabbit antibody directed against the tyrosine kinase domain of RET (Santa Cruz Biotechnology, catalogue item SC-167, Santa Cruz, California) was titered against control tissue culture NIH3T3 cells transfected with RET/PTC-1 (kindly provided by Dr. Sissy Jhiang) and nontransfected NIH3T3 cells. As the tyrosine kinase domain of RET is preserved in the translocation, this antibody is able to detect the rearranged version of RET in addition to proto-RET. The following protocol was used. The fixed cultures were rehydrated for 10 minutes in a 1:1 mixture of water and 95% flex alcohol (Richard-Allen), followed by three changes of TMS buffer (50 mmol/L Tris, pH 7.6, 5 mmol/L MgCl2, 150 mmol/L NaCl containing 10 μm/ml bovine serum albumin; Sigma). Blocking with 10% bovine serum plus blocking reagent from Elite Vectastain (Vector Laboratories, Burlingame, CA) diluted into TMS buffer was performed for 20 minutes at room temperature. Rabbit anti-RET antibody was diluted 1:80 into the blocking cocktail and incubated under a coverslip for 1 hour at 37°C. No antigen retrieval was used. Subsequent steps were performed with Vectastain Universal Quick Kit with the ABC method and with peroxidase-diaminobenzidine development as per the manufacturer's instructions (Vector Laboratories catalogue items PK-8800 and SK4100) except that two 10-minute incubations with diaminobenzidine (DAB) were used. Good discrimination between RET/PTC-positive and -negative NIH3T3 cells was obtained with this protocol, and alcohol-fixed morphology was well preserved. Positive immunostaining with this protocol was observed only in thyroid cultures retrovirally infected with RET/PTC. Thyroid cell cultures not infected with RET/PTC showed no staining, indicating that C cells are either not present or are not detected. A Papanicolaou stain was performed after DAB treatment, and cells were coverslipped with glycerol. For RAS immunostaining, a rat anti-RAS monoclonal antibody (Santa Cruz Biotechnology catalogue item SC-35) was titered against NIH3T3 cells overexpressing H-RAS (Ciras-2 cells, a gift of Jim Wright), using NIH3T3 parental cells as a negative control. The staining protocol was the same for RET with the following exceptions. Fixation did not include polyethylene glycol as this was found to diminish staining intensity. The anti-RAS antibody was diluted 1:100. A biotinylated goat anti-rat immunoglobulin (Vector Laboratories BA-9400) was used as a secondary antibody at recommended concentration. The ABC reagents from the Vector Elite System (catalogue item PK-6101) were used at recommended concentrations. Microscopic analysis was performed with a 100× oil-immersion objective with a 1.25 numerical aperture. Criteria were established by one cytopathologist that were likely to allow discimination between papillary thyroid carcinoma nuclei and normal thyroid nuclei. An irregular nuclear contour plus areas of open chromatin were used to define a cell as papillary thyroid carcinoma-like. For nuclear contour irregularity, it was found that it was easier to look for a smooth arc in nuclear contour rather than try to quantify the degree of irregularity. An arbitrary cut off of one-fourth or greater of the nuclear perimeter in a mid-nuclear plane with a smooth round contour was used to exclude the papillary thyroid carcinoma-like phenotype. Use of the criterion of a smooth contour for one-half the nuclear perimeter was more difficult to score due to nuclear undulations or occasional shallow irregularities of normal cells. To qualify as papillary thyroid carcinoma-like, cells also had to demonstrate a 2-μm-diameter intranuclear zone devoid of hematoxylin staining texture. This measurement was performed with a micrometer. To justify these criteria, a second cytopathologist, blinded to the nature of the study, was asked to identify differences between slides of cells retrovirally infected with RET/PTC compared with RAS and then later score the morphology using the criteria above. Both cytopathologists scored all nuclei in a random scan. Nuclei that could not be scored were counted as indicated, and these included mitotic cells and obscured, degenerated, or apoptotic cells. Nontransfected normal human thyroid cells in culture have nuclei that are flattened toward the substrate, with finely granular chromatin extending throughout the nucleoplasm, variable-sized nucleoli, and a smooth, round to oval nuclear contour. Figure 1, A, D, and F, shows normal thyroid epithelial cells (without brown anti-RET immunostain reaction product). The flattening of the nuclei is similar to what is seen in many other tissue culture cells and may also be seen in normal thyroid epitheliumin vivo. The chromatin of cultured normal thyroid cells has a slightly finer texture on average compared with thyroid cellsin vivo. Cultured thyroid cells expressing RET/PTC tend to disperse among the non-expressing cells on the culture plate, as demonstrated by RET immunostaining (brown DAB reaction product) (Figure 1A). They form occasional characteristic fenestrated colonies, previously described by low-magnification phase contrast microscopy.32Bond JA Wyllie FS Rowson J Radulescu A Wynford-Thomas D In vitro reconstruction of tumour initiation in a human epithelium.Oncogene. 1994; 9: 281-290PubMed Google Scholar The cytological features of these RET/PTC transfectants are different from the background cells (Figure 1, B, C, and E). 1) The majority of the nuclei of RET/PTC-expressing cells are not flattened toward the substrate, as illustrated in Figure 1, C–F. To show the nuclei of RET/PTC-expressing cells and non-expressing cells within the same field of view, the two sets of pictures in Figure 1, C and D), and Figure 1, E and F, are required to be in two different focal planes. 2) Many RET/PTC-expressing cells have irregular, unpredictable folds of the nuclear membrane (Figure 1, B, C, and E). 3) They tend to have open chromatin with relatively large areas showing no evident hematoxylin staining texture (Figure 1B, C, and E). A second cytopathologist, initially blinded to the cell types and genes studied, easily discerned a difference between RET/PTC-infected and H-RAS-infected cells and described this difference as follows: 1) presence of frequent nuclear contour irregularities, 2) open chromatin, and 3) variably prominent nucleoli of RET/PTC-infected compared with RAS-infected cells. To directly examine the relationship between RET/PTC expression and altered nuclear morphology, cells were scored for the presence of the papillary thyroid carcinoma-like morphology (defined in Materials and Methods) and RET/PTC expression. Normal human thyroid epithelial cells were scored after having been infected 10 days previously with RET/PTC, grown without G418 selection, and immunostained for RET/PTC. Cytopathologist 1 determined the proportions of papillary thyroid carcinoma-like cells and RET/PTC-immunostained cells from a random population of 2000 intact cells. The results (Table 1) show that 42% of RET/PTC-expressing cells show a papillary thyroid carcinoma-like phenotype compared with only 2.5% of non-RET/PTC-expressing cells (P ≪ 0.0001 by χ2Rosai J Carcangiu ML Delellis RA Tumors of the Thyroid Gland, Fascicle 5, third series.in: Rosai J Sobin LH Armed Forces Institute of Pathology, Washington, DC1992Google Scholar analysis). A second independent cytopathologist, given the same criteria for scoring, but blinded to the cell type and gene studied, scored 500 immunostained (RET/PTC-expressing) and 500 non-immunostained cells. The second cytopathologist included all of the nuclei encountered in a random scan, even if they were non-scorable. The results (Table 1)independently show RET/PTC expression to be highly associated with a papillary thyroid carcinoma-like phenotype with 52% of the RET/PTC-expressing cells showing a papillary thyroid carcinoma-like phenotype compared with 6% of the non-expressing cells (a maximum of 9% if every non-scorable nucleus were to have had papillary thyroid carcinoma-like features; P ≪ 0.0001).Table 1Association of RET/PTC Expression with an Irregular Nuclear Contour and EuchromatinObserver 1Observer 2RET/PTC immunostained cellsNo immunostaining500 RET/PTC immunostained cells500 non-immunostained cellsPTC-like phenotype254825732Not PTC-like341893*P < 0.0001 by χ2 analysis.232453Non-scorable1115A PTC-like phenotype was defined as an irregular nuclear contour over more than three-fourths of the nuclear perimeter and a 2-μm-diameter area of absent hematoxylin staining texture. Any nucleus that was degenerated, apoptotic, mitotic, or obscured was counted as non-scorable.* P < 0.0001 by χ2 analysis. Open table in a new tab A PTC-like phenotype was defined as an irregular nuclear contour over more than three-fourths of the nuclear perimeter and a 2-μm-diameter area of absent hematoxylin staining texture. Any nucleus that was degenerated, apoptotic, mitotic, or obscured was counted as non-scorable. The immunostaining of RET/PTC is diffuse throughout the cytoplasm with no apparent accentuation of the perinuclear or other sub-cytoplasmic domain. Rare intranuclear cytoplasmic inclusions were observed (Figure 1B), and only in RET/PTC-infected cells. Primary normal human thyroid cells transfected with H-RAS tend to grow contiguously to form a discrete colony that pushes back the surrounding normal monolayer (Figure 2A), as previously described by phase contrast microscopy.32Bond JA Wyllie FS Rowson J Radulescu A Wynford-Thomas D In vitro reconstruction of tumour initiation in a human epithelium.Oncogene. 1994; 9: 281-290PubMed Google Scholar The nuclei of these cells show a slight coarsening of chromatin and are more spherical than those of nontransfecte
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