The previous studies of p16^INK4a, pRb, p53, and Ki-67 expression suggested that these markers may be preferentially expressed in cervical neoplasms. The purpose of this study was to assess the expression and the clinical significance of p16^INK4a, pRb, p53, and Ki-67 proteins in cervical lesions. We obtained 106 cases with various categories of cervical squamous mucosa, including squamous cell carcinoma (n=35), cervical intraepithelial neoplasia (CIN) II/III (n=26), CIN I (n=10), squamous metaplasia (n=15), and normal squamous mucosa (n=20). Immunohistochemical staining was performed for p16INK4a, pRb, p53, and Ki-67 proteins in formalin-fixed and paraffin-embedded tissue sections of the uterine cervix. Evaluation of immunohistochemical staining was based on the frequencies of expression and the mean immunoreactivity scores (IS) in each diagnostic category. p16INK4a positive sotaining was observed in 26 of 35 cases (74.3%) of squamous cell carcinoma, in 16 of 26 cases (61.5%) of CIN II/III, in six of 10 cases (60%) of CIN I, in nine of 15 cases (60%) of squamous metaplasia, and negative in normal squamous mucosa. pRb expression was detected in all diagnostic categories; however, the proportion of pRb positive cells was relatively decreased in CIN II/III (38.5%) and squamous cell carcinomas (51.4%), compared to normal squamous epithelium (90%) and squamous metaplasia (73.3%). No significant differences in expression of p53 were observed in any diagnostic categories. Ki-67 expression was increased in squamous cell carcinoma (37.1%), CIN II/ III (42.3%), and CIN I (40%), but negative in squamous metaplasia and normal mucosa. In 35 cases of squamous cell carcinomas, multivariate analysis revealed no differences in p^INK4a, pRb, p53, and Ki-67 expression according to the age of the patient, lymph node metastasis and clinical stage. In conclusion, the combined use of p16^INK4a and Ki-67 immunoreactivity could improve the diagnostic specificity of squamous cell carcinoma of the uterine cervix.

Retinoic acid plays an important role in the regulation of cell growth and differentiation. In our present study, we evaluated the effects of all-trans retinoic acid (RA) on cell proliferation and on the cell cycle regulation of human gingival fibroblasts (HGFs). Cell proliferation was assessed using the MTT assay. Cell cycle analysis was performed by flow cytometry, and cell cycle regulatory proteins were determined by western blot. Cell proliferation was increased in the presence of a 0.1 nM to 1μM RA dose range, and maximal growth stimulation was observed in cells exposed to 1 nM of RA. Exposure of HGFs to 1 nM of RA resulted in an augmented cell cycle progression. To elucidate the molecular mechanisms underlying cell cycle regulation by RA, we measured the intracellular levels of major cell cycle regulatory proteins. The levels of cyclin E and cyclin-dependent kinase (CDK) 2 were found to be increased in HGFs following 1 nM of RA treatment. However, the levels of cyclin D, CDK 4, and CDK 6 were unchanged under these conditions. Also after exposure to 1 nM of RA, the protein levels of p21 WAF1/CIP1 and p16 INK4A were decreased in HGFs compared with the control group, but the levels of p53 and pRb were similar between treated and untreated cells. These results suggest that RA increases cell proliferation and cell cycle progression in HGFs via increased cellular levels of cyclin E and CDK 2, and decreased cellular levels of p21 WAF1/CIP1 and p16 INK4A.

The p16 gene encodes an inhibitor of the cyclin-dependent kinase, which inactivates cyclin-dependent kinase and contro1s the cell cycle progression, The 10ss of p16 expression or overexpression has been reported in many kinds of tumors, Both p16 and PCNA regu1ates cell cycle progression at the Gl/8 checkpoint, Although many researches about the p16 expression in ora1 cancer have been carried out, there are few studies about the corre1ation between p16 ex pression and pro1iferation of ora1 cancer cells The object of this study was to eva1uate the avai1ability of p16 as ear1y diagnostic factor and prognostic factor through corre1atión ana1ysis of p16 expression in ora1 squamous cell carcinoma and its re1ation to PCNA index and clinicopatho1ogic factors 80 we investigated p16 immunohistochemica1 expression of 83 ora1 squmaous cell carcinomas, and obtained the resu1ts as followed, 18 out of the 83 cases(21, 69%) showed p16 positive and 65 samp1es(78,31%) showed p16 negative, Whi1e the mean va1ue of PCNA indices of p16 positive cases was 65,94 ::t 18,32, that of PCNA indices at p16 negati ve ones 54,79 ::t 18, 39, This difference between them showed statistica1 sígnificance, (P=O, 030) p16 positive group was 12/60(20, 0%) of well differentiated tumors and p16 negative group was 6/23(16, 1%) of moderate1y or poor1y differentiated tumors, This difference did not show statistica1 significance. (P=O. 372) From the resu1ts above, it was suggested p16 expression is re1ated to PCNA index in ora1 squamous cell carcinomas.

p16INK4A and p15INK4B tumor suppressor genes are frequently altered in various human tumors. Hypermethylation of the promoter region of p16INK4A and p15INK4B seem to be the major mechanism of inactivation. To determine whether the change in p16INK4A and p15INK4B methylation status occur in oral squamous cell carcinomas (OSCCs) and benign oral epithelial hyperplasias, we analyzed 46 OSCCs and 20 benign oral epithelial hyperplasias by methylation-specific PCR. We also analyzed a subset of the samples for p16INK4A and p15INK4B protein expression by immunohistochemistry. The promoter region of p15INK4B was hypermethylated in 13 specimens of the 15 finally analyzed OSCCs and three specimens of the five analyzed benign oral epithelial hyperplasias. By immunohistochemical analysis, we confirmed the loss of p15INK4B expression of all hypermethylated specimens. The promoter region of p16INK4A was amplified by both an unmethylated- and a methylated-specific primers in just one OSCCs. The remaining specimens including 11 OSCCs and four benign oral epithelial hyperplasias were normally methylated. By immunohistochemistry, we analyzed the loss of p16INK4A expression in seven specimens of the 12 OSCCs and two specimens of the four benign oral epithelial hyperplasias. Except for one OSCC, however, all specimens showing loss of expression were normally methylated. These results suggest that loss of p16INK4A and p15INK4B protein expression play an important role in the development of both OSCCs and benign oral epithelial hyperplasias.