Cell cycle proteins and the development of oral squamous cell carcinoma

Abstract

Expression of cell cycle regulatory proteins was evaluated in premalignant and malignant oral epithelial lesions, to test the hypothesis that protein regulation of the cell cycle may be altered in the development of oral squamous cell carcinoma. Archived paraffin-embedded specimens (n=90) from 25 patients with recurrent or persistent lesions were evaluated in immunohistochemically stained sections for cell cycle regulatory proteins p53, Rb, Cyclin D1, p27, and p21. The cell cycle was also evaluated by expression of nuclear protein Ki 67. Sections were graded semiquantitatively using a 0 − 3 + scale to indicate the percentage of positively stained cells. The initial histologic diagnosis for 17/25 patients was either focal keratosis, mild dysplasia, or moderate dysplasia; the initial diagnosis for the remaining eight patients ranged from severe dysplasia to moderately differentiated squamous cell carcinoma. Thirty-three of 90 specimens showed positive p53 expression, 11 of which were dysplasias. Eighty-nine of 90 specimens, from all stages of disease, showed positive Rb expression. Twenty-three of 90 specimens showed positive Cyclin D1 expression, typically in the later stages (carcinoma) of a patient's disease. Eighty-four of 90 specimens showed positive p21 expression; while 55 of 90 specimens were positive for p27. In control mucosa, p27 was highly expressed, while Rb and p21 proteins were expressed at relatively low levels; p53 and Cyclin D1 proteins were largely absent. Generally, staining of p53, Rb, p21, and Ki 67 increased with time in serial biopsies, while p27 showed decreased staining with disease progression. These data show that cell cycle regulatory proteins are altered in both premalignant and malignant disease, and that protein phenotypes are heterogeneous. P53 expression is seen early, and Cyclin D1 expression is seen late in the development of oral premalignant and malignant disease. Expression of p53, Rb, p21 and Ki67 increased, while p27 decreased, with disease progression.

Introduction

Progression through the cell cycle is regulated by a variety of proteins that play an important role in allowing the correct order and timing of events involved in chromosomal repair, duplication, and separation. Cyclin dependent kinases (CDK) and their regulatory partners, cyclins, form heterodimeric protein complexes that appear and degrade during predetermined steps in the cell cycle1, 2, 3. These protein complexes allow for orderly progression of the cell cycle, and also act to phosphorylate fundamental cell cycle proteins, such as retinoblastoma protein (pRb)4, 5, 6, 7.

Phosphorylation of pRb during the G1 phase of the cell cycle allows for transition from G1 to S-phase through release of transcription factors important in the induction of S-phase8, 9, 10, 11, 12. Many cyclins and CDK's have been identified, as well as three members of the pRb family13, 14, 15, 16, 17. These proteins have been mapped according to their appearance in specific periods of the cell cycle, as well as to their protein binding partners[18]. Rb mutations and altered patterns of pRb expression have been demonstrated in a wide variety of human tumors, including oral cavity and head and neck squamous cell carcinoma19, 20, 21, 22. Cyclin D1 is an unstable protein essential for G1 progression, through complex formation with CDK4, and pRb binding[8]. Cyclin D1 gene amplification and protein over expression have been found in both breast cancer and head and neck squamous cell cancers23, 24, 25, 26, 27, 28.

In addition to regulation by cyclin binding and phosphorylation, cyclin dependent kinases are regulated by specific proteins called cyclin dependent kinase inhibitors (CDKI)29, 30. CDKI's in mammals fall into two general families; the p21 family (p21^Cip1/WAF1, p27^Kip1 , and p57^Kip2), and the INK4 family (p15^INK4b, p16^INK4a, p18^INK4c, and p19^INK4d)31, 32, 33, 34, 35, 36, 37, 38, 39, 40. The CDK inhibitor p16 binds to CDK4 and CDK6, inhibits phosphorylation of Rb, leading to G1 arrest8, 41. Loss of p16 function has been demonstrated in a wide variety of human tumors, including oral and esophageal cancer42, 43, 44, 45, 46, 47. p21 acts on multiple cyclin/CDK complexes to effect arrest of the G1/S-phase of the cell cycle31, 48, 49, 50, 51, 52. This protein is frequently expressed in epithelial cells, and may play a role in maintaining cells in a non-mitotic condition53, 54. p21 has been shown to be transcriptionally regulated by the tumor suppressor protein p53, providing an important possible explanation for p53 mediated cell cycle G1 arrest31, 50, 55, 56. p21 induction can also be accomplished by a p53 independent pathway57, 58, 59.

The role of wild type p53 protein in inducing cell cycle arrest to allow for repair of damaged DNA has been well characterized60, 61, 62. Under certain conditions, (e.g., DNA damage, withdrawal of growth factors) p53 activation can lead to apoptosis. Studies in cultured cells63, 64have shown that p53 protein can trigger arrest in either the G1 or G2 phase of the cell cycle. Loss of p53 function here could allow the accumulation of mutations that represent the rate limiting steps in tumor progression. Loss of p53 function has been demonstrated in about one half of all human cancers, including oral squamous cell carcinoma[60].

The CKI p27^Kip1 shares sequence homology with p21, and acts as a p53 independent negative cell cycle regulator involved in G1 arrest32, 33, 65. This protein also associates with several cyclin-CDK complexes, resulting in loss of their activity, and failure to phosphorylate Rb. The p27 gene has not been associated with mutations in human tumors, although underexpression has been demonstrated in oral cancers66, 67.

The purpose of this investigation was to evaluate the expression of cell cycle regulatory proteins (Cyclin D1, Rb, p21, p27, p53) in sequential pre-malignant (focal keratosis, dysplasia,) and malignant (carcinoma in situ, squamous cell carcinoma) lesions arising in the oral cavity to test the hypothesis that protein regulation of the cell cycle is altered in the development of oral squamous cell carcinoma. The proliferative index was also assessed by the evaluation of expression of nuclear protein Ki67. Cases selected were archived paraffin-embedded specimens from patients with two or more sequential oral biopsies with diagnoses of focal keratosis, dysplasia of all grades, carcinoma in situ, or squamous cell carcinoma. Comparisons were made between immunohistochemical staining patterns and disease progression.