Abstract
Somatic mutations in the retinoblastoma-1 gene (RB1) and loss of RB1 protein function have been implicated in a number of human malignancies, but the role of RB1 gene and protein abnormalities in ductal pancreatic cancer (DPCA) is virtually unknown. We therefore analyzed expression of the RB1 protein immunohistochemically and/or by western blotting in a total of 54 sporadic and eight familial cases of archival and frozen DPCA and in 18 pancreatic carcinoma cell lines by using the antibodies RB-WL-1, 84-B3-1, and PMG3-245. Mutations in the RB1 promoter region and exons 13-21 of the RB1 gene were likewise examined by single-strand conformation polymorphism (SSCP) analyses and DNA sequencing of genomic DNA from 30 microdissected primary pancreatic tumors and the pancreatic carcinoma cell lines. Moreover, amplification and expression of a major regulatory component of RB1 function, cyclin D1, were assessed by Southern and immunohistochemical analyses, respectively. The DPCAs were heterogeneous in both the intensity of RB1 nuclear staining and the percentage of immunoreactive cells. The tumors often had areas where RB1 staining was weak or absent adjacent to normal pancreatic tissue; however, only two of 32 archival cases and one of 30 frozen cases of DPCA completely lacked RB1 nuclear staining. Immunohistochemical and western blot analyses of 18 pancreatic carcinoma cell lines demonstrated the absence of RB1 expression in only two cell lines, Capan-1 and GCP-1. Analyses of the RB1 gene and promoter region by SSCP and DNA sequencing largely confirmed the immunochemical findings. Three of 30 primary carcinomas had abnormalities revealed by SSCP analyses. In one case a single base-pair deletion was confirmed in exon 18 and resulted in premature termination and the absence of detectable RB1 protein. A second case had a TAC → TTC missense mutation in exon 13. The third primary carcinoma could not be reliably sequenced because it had a low percentage of epithelial cells. The cyclin D1 gene was not amplified in any of the primary pancreatic tumors or cell lines examined. These immunochemical and molecular analyses of the RB1 tumor suppressor gene and cyclin D1 proto-oncogene in a large series of human pancreatic cancers and cell lines indicate that RB1 and cyclin D1 alterations occur during the development of some human DPCAs. Nevertheless, it is probable that alterations in cell-cycle regulation in DPCAs more frequently involve pathways other than those involving RB1 and cyclin D1.