A novel, nuclear pore-associated, widely distributed molecule overexpressed in oncogenesis and development

Abstract

Nuclear pore complexes are large, elaborate macromolecular structures that mediate the bidirectional nucleocytoplasmic traffic. In vertebrates, nuclear pore complexes comprise 50 to 100 proteins termed nucleoporins (Nup). An 88-kd nucleoporin (Nup88) has been recently cloned and characterized, and found to be associated in a dynamic subcomplex with the oncogenic nucleoporin CAN/Nup 214. We have produced a polyclonal antiserum to Nup88, and found that it immunoreacts convincingly in conventional tissue sections of 214 samples of malignant tumors of many types. All carcinomas were stained irrespective of site or line of differentiation; the majority of cases reacted strongly and extensively. In situ carcinomas and highly dysplastic epithelia were similarly reactive. Samples of malignant mesotheliomas, gliomas, sarcomas, and lymphoreticular tumors were also stained. Substantial reactions were also found in certain fetal tissues. Focal reactions were noted in some reactive-proliferative processes. Most benign epithelial and mesenchymal tumors and hyperplasias, and normal adult tissues reacted weakly and sporadically or not at all. Immunoblot analysis of selected samples strongly corroborated those findings. If further substantiated, our findings indicate that Nup88 could be regarded as a selective yet broadly based proliferation marker of potential significance in the histological evaluation and diagnosis of malignant transformation. Its ready applicability on conventional paraffin sections and on cytological preparations may broaden its clinical and investigative significance.

Figure 1.

All samples were immunostained with polyclonal antiserum to Nup88; the chromogen is DAB. a: Stomach adenocarcinoma; the prominent mucosa (top) is not reactive, whereas the carcinoma invading the submucosa (bottom right) is clearly stained. Original magnification, ×60. b: Stomach adenocarcinoma; higher magnification of tumor in a shows diffuse and strong staining of the malignant glands. Original magnification, ×480. c: Stomach with extensive intestinal metaplasia; a strongly and diffusely reactive in situ carcinoma is evident. Original magnification, ×180. d: Colon adenocarcinoma; the normal mucosa on the left is negative whereas the basal aspect of the glands of a villous adenoma are moderately reactive (arrow). The overt carcinoma invading the submucosa is richly stained. Original magnification, ×120. e: Colon adenocarcinoma; higher magnification of tumor in d shows diffuse, strong, and granular reaction of malignant glands. Original magnification, ×620. f: Colon; neuroendocrine carcinoma showing portions of characteristically organoid clusters diffusely reactive for Nup88. Original magnification, ×480. g: Liver; cirrhotic septum in the vicinity of the carcinoma depicted in h. Note focal staining in proliferating bile ductules (arrows) amid nonreactive fibroconnective tissue. Original magnification, ×180. h: Liver; hepatocellular carcinoma; higher magnification shows strong, extensive, and granular reaction. Original magnification, ×620. i: Pancreas; adenocarcinoma showing strong Nup88 staining of malignant glands. Original magnification, ×420. j: Breast; infiltrating ductal carcinoma showing strongly Nup88 reactive clusters. Original magnification, ×420. k: Breast; fibrocystic changes; hyperplastic and cystic ducts are nonreactive. Original magnification, ×120. l: Breast; intraductal carcinoma, cribriform variant. The staining is diffuse but is best appreciated toward the periphery of the duct (arrows). Original magnification, ×120. m: Lung; well-differentiated squamous carcinoma; part of neoplastic cluster showing strong staining particularly of the peripheral cells; the central, more mature cells approaching pearl formation show fewer reactive granules. Original magnification, ×620. n: Lung; moderately differentiated adenocarcinoma; note strong and diffuse reaction. Original magnification, ×480. o: Bronchial carcinoid. Note moderately but diffusely reactive cell ribbons and clusters amid negative stroma. Original magnification, ×180.

Figure 2.

The same antiserum was used in all samples. The chromogen was DAB except for n for which alkaline phosphatase (red) was used. a: Ovary; moderately differentiated papillary serous carcinoma; note rich, diffuse reaction of neoplastic papillae. Original magnification, ×480. b: Endometrium; adenocarcinoma, well differentiated, showing strong and diffuse Nup88 reactivity. Original magnification, ×480. c: Endometrium; cystic hyperplasia; no reaction is evident. Original magnification, ×180. d: Prostate; adenocarcinoma, Gleason 4 + 5; intense reaction involving the majority of malignant cells is seen. Original magnification, ×420. e: Prostate; periphery of carcinoma shown in d. Small clusters of malignant cells are strongly stained; note also the intense reaction of neoplastic cells around and within a nerve. Original magnification, ×540. f: Prostate showing dilated, hyperplastic, and atrophic glands; no Nup88 reaction is seen. Original magnification, ×180. g: Kidney; clear cell carcinoma. A strong and extensive reaction is evident. Original magnification, ×480. h: Soft tissue; malignant fibrous histiocytoma; a weak to moderate but extensive reaction involves most neoplastic cells. Original magnification, ×480. i: Leiomyosarcoma metastatic to the lung (see Figure 3 ▶ , immunoblot B5). Strong reactivity is noted particularly in the bizarre giant cells. Original magnification, ×620. j: Lymph node; large cell lymphoma diffusely reactive for Nup88. Original magnification, ×160. k: Pleura; malignant papillary mesothelioma richly and diffusely reactive for Nup88. Original magnification, ×480. l: Abdomen; benign cystic mesothelioma; no reaction is seen. Original magnification, ×480. m: Brain; glioblastoma multiforme; a rich and extensive reaction is noted. Original magnification, ×480. n: Skin; malignant melanoma immunostained for Nup88 with alkaline phosphatase as chromogen (red). A rich and extensive reaction is evident. Original magnification, ×480. o: Fetal lung (20 weeks, 360 g). A moderate immunoreaction involves the primitive air spaces whereas the stroma appears nonreactive. Original magnification, ×320.

Figure 3.

Immunoblot analysis of Nup88 content in neoplastic, hyperplastic, and normal tissues. Total protein extracts of colon (A), lung (B), breast (C), and ovary (D) were prepared as described in Materials and Methods with the polyclonal antiserum to Nup 88. A: Colon. Lanes 1, 3, 4, and 5 represent adenocarcinomas; lane 2 represents also an adenocarcinoma but showing extensive necrosis; lane 6 is the negative control consisting of normal human lymphocytes whereas lane 7 is the positive control representing an ovarian carcinoma known to express abundant Nup88 from previous studies. B: Lung. Lane 1 represents the bronchial carcinoid depicted in Figure 1o ▶ . Lanes 2 and 4 represent an adenocarcinoma and a squamous carcinoma, respectively, whereas lane 3 is a poorly differentiated squamous carcinoma treated with chemotherapy and radiotherapy before excision. The strongly reactive lane 5 represents a metastatic leiomyosarcoma shown in Figure 2i ▶ ; lane 6 represents hyperplastic bronchi in the vicinity of but not involved by a carcinoma. Negative control in lane 7 as in A. C: Breast. Lane 1 represents typical, benign fibrocystic disease depicted in Figure 1k ▶ . Lanes 2 and 3 represent fibroadenomas; lanes 4 and 5 represent infiltrating ductal carcinomas whereas lane 6 is an infiltrating lobular carcinoma. Lane 7 is a normal control adult female breast and lane 8 is a known, positive control ovarian carcinoma. D: Ovary. Lane 1 represents a benign mucinous cystadenoma; lanes 2 and 3 represent papillary serous carcinomas, and lane 4 is a metastasis from a gastric signet-ring cell carcinoma. Lane 5 shows a surprisingly low signal given by an endometrioid carcinoma possibly reflecting sampling discrepancies. Lanes 6, 7, and 8 are negative controls and lane 9 is the positive counterpart.