Effect of p27 on cyclin E-CDK2 complex. p27 binds to cyclin E-CDK2 complexes and prevent its activation. Although some p27 is present in proliferating cells, it is sequestered and unavailable to interact with cyclin E-CDK2. p27 can be regulated by the same enzyme it targets for inhibition by becoming a cyclin E-CDK2 substrate, leading to its phosphorylation and proteolysis by the ubiquitin-proteasome pathway. Phosphorylation of retinoblastoma (Rb) protein leads to G₁ to S progression.

Schematic view of mammalian cyclin-dependent kinase (CDK) inhibitors and cyclin-CDK complexes in the cell cycle. Members of the INK4 group (p15/INK4B, p16/INK4A, p18/INK4C, and p19/INK4D) and the Cip/Kip group (p21, WAF/CIPI, p27/Kip1, and p57/Kip2) have inhibitory roles in G₁ to S progression.

Schematic model showing the influence of p27, other CDKIs and cyclins on the development of endocrine cell hyperplasia and neoplasia. Dysregulation of various CDKIs, including p27, p16, p21, and p57 and of the D-type cyclins have been observed in various proliferating endocrine tissues (see text). Dysregulation of CDKIs and cyclins leading to increased cell proliferation in adult endocrine tissues increases the likelihood of developing genetic alterations resulting in tumor development.

Immunohistochemical staining for p27 expression in normal and neoplastic tissues. A: Lymph node tissue with p27 staining on left showing strong immunoreactivity in quiescent cells and less staining in proliferating cells. The germinal center (GC) with proliferating cells had few cells positive for p27. Staining with a Ki67 antibody MIB-1, which recognizes proliferating cells, showed the opposite pattern of staining compared with p27 with strong staining of most cells in the GC. Magnification, ×200. B: There is selective localization of p27 in normal anterior pituitary cells. Immunostaining of normal anterior pituitary for p27 (nuclear purple staining) and for thyroid stimulating hormone (TSH; brown cytoplasmic staining) shows localization of p27 in some TSH cells (arrows). Magnification, ×300. C: Normal and neoplastic breast tissue with strong staining for p27 in normal mammary ducts whereas the invasive carcinoma cells are mostly negative. Magnification, ×300. D: Normal and neoplastic prostate tissue showing strong staining for p27 in normal prostatic ducts while the invasive carcinoma cells are weakly positive or negative. Magnification, ×300. E: Parathyroid tissue showing strong nuclear staining for p27 in the normal cells on the left whereas the adenoma on the right stains weakly, indicating low expression of p27. Magnification, ×250. F: The normal thyroid on the left shows strong nuclear staining for p27 in the follicular cells. The papillary carcinoma on the right shows low expression of p27 protein. Magnification, ×250.

Analysis of the effect of inhibition of the chymotryptic site of the ubiquitin-proteasome pathway on cellular p27. The HP75 pituitary cell line (produced in our laboratory) was treated with the peptide-aldehyde N-acetyl-leucinyl-leucinyl-norleucinal-H (LLnL), an inhibitor of the chymotryptic site on the proteasome, or with the cysteine protease inhibitor l-trans-expoxysuccinic acid (E64) as a control for 16 hours in culture. The cells were homogenized and analyzed for p27 by Western blotting using a monoclonal antibody (Transduction Laboratories, Lexington, KY) and enhanced chemiluminescence (Amersham, Arlington Heights, IL). The samples include the following: lane 1, control cells with culture media only; lane 2, cells treated with dimethylsulfoxide (100-μl volume equivalent to the LLNL and E64 vehicle volume); Lane 3, 50 μmol/L LLnL; lane 4, 100 μmol/L LLnL; lane 5, 50 μmol/L E64; lane 6, 100 μmol/L E64; lane 7, HeLa cells used as a p27-positive control. LLnL, but not E64, increased p27 protein in the HP75 cells. β-Actin was used to normalize for protein loading. The graph on the right was generated by densitometric analysis of the film. These results indicate that the ubiquitin-proteasome pathway is one of the mechanisms regulating the expression of p27 protein in pituitary tumor cells.