Association of cyclin D1, p16 and retinoblastoma protein expressions with prognosis and metastasis of gallbladder carcinoma

Abstract

AIM: To investigate the role of cyclin D1, p16 and retinoblastoma in cancerous process of gallbladder carcinomas and to assess the relation between cyclin D1, p16, Rb and the biological characteristics of gallbladder carcinoma.

METHODS: Forty-one gallbladder carcinoma, 7 gallbladder adenoma and 14 chronic cholecystitis specimens were immunohistochemically and histopathologically investigated for the relation of cyclin D1, p16 and Rb with Nevin staging and pathologic grading.

RESULTS: The expression rates of abnormal cyclin D1 in gallbladder carcinoma (68.3%)and gallbladder adenoma(57.1%) were significantly higher than those in chronic cholecystitis (7.1%) (P<0.05). No significant difference was found both among the pathological grades G₁, G₂ and G₃ and among Nevin stagings S₁-S₂, S₃ and S₄-S₅ of gallbladder carcinoma. The positive rates of p16 (48.8%) and Rb (58.5%) in gallbladder carcinoma were significantly lower compared to those in adenoma (100.0%) and cholecystitis (100.0%) (P<0.05). The positive rates of p16 and Rb in Nevin stagings S₁-S₂ (80.0% and 90.0%) and S₃ (46.2% and 61.5%) gallbladder carcinomas were significantly higher than those in S₄-S₅ (33.3% and 38.8%) (P<0.05), and those in pathologic grades G₁ (54.5% and 81.8%) and G₂ (50.0% and 62.5%) gallbladder carcinoma were significantly higher than those in G₃ (28.6% and 35.7%) (P<0.05). The protein expression of p16 and Rb had a negative-correlation in gallbladder carcinoma (r = -0.2993, P<0.05), and this negative-correlation was correlated with Nevin staging (P<0.05). Moreover, the protein expression of p16 and cyclin D1 had a negative-correlation in gallbladder carcinoma (r = -0.9417, P<0.05).

CONCLUSION: Cyclin D1 may play a role in the early stage of gallbladder carcinoma. Mutation of p16 and Rb genes might be correlated with progression of gallbladder carcinoma. Analysis of p16 and Rb can estimate the prognosis of gallbladder carcinoma. Expression of p16 and Rb may be correlated with Nevin staging and pathologic grading in gallbladder carcinoma.

Key Words: Gallbladder carcinoma; Cyclin D1; p16 protein; Retinoblastoma protein; Tumor metastasis

INTRODUCTION

Primary carcinoma of gallbladder is a very lethal malignant tumor because of its early metastasis, strong invasion and poor prognosis. It is very important to estimate the malignant degree and invasion tendency in order to guide clinical diagnosis and treatment of gallbladder carcinoma[1,2]. Cyclin D1 is considered as an oncogene and can promote progression of the cell cycle to S by cyclin D-dependent kinases (CDK4/CDK6)-mediated phosphorylation of the retinoblastoma (Rb) protein[3,4]. The activities of CDK4/CDK6 are constrained by p16[4,5]. The onset and progression of gallbladder carcinoma are accompanied with multiple genetic changes that result in qualitative and quantitative alterations in individual gene expression[3-6]. By immunohistochemical methods, we analyzed cyclin D1, p16 and Rb expression levels in gallbladder carcinomas, adenomas and cholecystitis to evaluate their relationships with the pathogenesis, development and metastasis of gallbladder carcinoma.

MATERIALS AND METHODS

Materials

Sixty-two randomly chosen cholecystectomy specimens included 41 gallbladder carcinomas, 7 gallbladder adenomas and 14 chronic cholecystitis. The Nevin staging of gallbladder carcinoma, and pathologic grading of all cases were determined based on clinical materials. Ten cases were determined as S₁-S₂, 13 cases as S3 and 18 cases as S₄-S₅, by Nevin staging. Eleven cases were determined as G₁, 16 cases as G₂ and 14 cases as G₃ by pathologic grading. The polyclonal p16 antibody was rabbit antiserum against human p16 protein (Dako co, USA). The polyclonal Rb antibody was rabbit antiserum against human Rb protein (Santa Cruz, USA). The monoclonal cyclin D1 antibody was mouse antiserum against human cyclin D1 protein (Santa Cruz, USA).

Methods

Tissue specimens of gallbladder carcinoma were formalin-fixed and paraffin-embedded, and cut into 5 μm thick sections for staining. The working concentrations of p16, cyclin D1 and Rb antibodies were 1:40, 1:80 and 1:100, respectively. Paraffin-embedded sections of gallbladder carcinoma tissue were dewaxed and dehydrated with ethanol. The sections were incubated with 3 mL/L H2O2-methyl alcohol for 30 min. The slides were incubated overnight with appropriate dilutions of monoclonal antibodies/polyclonal antibodies (McAbs/PcAbs) in PBS (pH 7.8) at 4 °C. After several washing steps, the reactivity was visualized using streptavidin/horseradish peroxidase-conjugated horse anti-mouse or goat anti-rabbit immunoglobulin (Zymed Co) diluted at 1:500 in PBS. Diaminobenzidine and hydrogen peroxide were used as substrates. Control slides were incubated with pre-immune sera or PBS. The protein expression was then scored arbitrarily according to the following scales: -, <25% positive cells; +, 25-50% positive cells; ++, >50-75% positive cells; and +++, >75% positive cells. All steps were carried out at room temperature. All reagents were equilibrated at room temperature.

Statistical analysis

Two×two contingency table, chi-square test, and correlation analysis were performed. SPSS 8.0 statistical software was used for calculation. P<0.05 was considered statistically significant.

RESULTS

Expression of p16, Rb and cyclin D1 gene in gallbladder carcinoma

The positive staining for p16 and Rb gene expressed as brown granules, was mainly located in nuclei of tumor cells, partly in cytoplasms (Figure 1: A, B). The positive staining for cyclin D1 gene expressed as brown granules, was distributed mainly in cell nuclei (Figure 1C).

Figure 1 Immunohistochemical staining for Rb, p16 and cyclin D1 (×400). A: Rb-positive expression; B: p16-positive expression; C: cyclin D1-positive expression.

The positive expression rates of cyclin D1, p16 and Rb are shown in Table 1. The abnormal cyclin D1 expression rate in gallbladder carcinoma and gallbladder adenoma was significantly higher than that in chronic cholecystitis (P<0.05). No significant difference was found between gallbladder carcinoma and adenoma, and in pathologic grades G₁, G₂ and G₃, and in Nevin stagings S₁-S₂, S₃ and S₄-S₅ of gallbladder carcinoma.

Table 1 Positive expression rates of Rb, p16 and cyclin D1.

Disease	n	Rb		Cyclin D1		p16
		+	%	+	%	+	%
Gallbladder carcinoma	41	24	58.7	28	68.3	20	48.8
Adenoma	7	7	100	4	57.1	7	100
Cholecystitis	14	14	100	1	7.1	13	92.8

The positive expression rates of p16 and Rb in gallbladder carcinomas were significantly higher than those in adenomas and cholecystitis (P<0.05). The positive expression rates of p16 and Rb in Nevin stagings S₁-S₂ and S₃ of gallbladder carcinoma were significantly higher than those in S₄-S₅ (P<0.05). There was also a significant difference (P<0.05) in p16 and Rb expression between pathologic grades G₁ and G₃ of gallbladder carcinoma (P<0.05) (Table 2).

Table 2 Correlation between expression rates of Rb, p16 and cyclin D1, and carcinoma staging.

Stages		n	Rb		Cyclin D1		p16
			+	%	+	%	+	%
Pathologic grading	G1	11	9	81.8	8	72.7	6	54.5
	G2	16	10	62.5	11	68.8	8	50
	G3	14	5	35.7	11	78.6	4	28.6
Nevin staging	S1-S2	10	9	90	6	60	8	80
	S3	13	8	61.5	8	61.5	6	46.2
	S4-S5	18	7	38.8	14	77.8	6	33.3

Correlation among Rb, cyclin D1 and p16 protein expression in gallbladder carcinoma

Among the 20 cases of p16-positive gallbladder cancer, 8 were Rb positive. Of the 24 cases of Rb-positive gallbladder cancer, 8 were p16 positive. Tumor suppressor gene p16 was correlated with Rb (χ² = 5.53, P<0.05; r = -0.2993, P<0.05). There was a negative correlation between p16 and cyclin D1 (χ² = 6.03, P<0.05; r = -0.9417, P<0.05), but no correlation between Rb and cyclin D1 (χ² = 1.20) (Table 3).

Table 3 Relationship between expressions of Rb, cyclin D1 and p16 in gallbladder carcinoma.

p16	Rb		Cyclin D1		Total
	+	-	+	-
+	8	12	10	10	20
-	16	5	18	3	21
Total	24	17	28	13	41

DISCUSSION

It has been accepted that there is a restriction point in cell-cycle progression, the restriction point of G1-S is important. Rb, p16 and cyclin D1 are major restriction factors in the restriction point. The cyclin D1/p16/Rb pathway plays a critical role in tumorigenesis and each component of this pathway may be affected by various malignancies[7-11]. Cell-cycle progression is normally regulated by cyclins and cyclin inhibiting proteins. Progression of cells from G₁ to S phase is regulated via pRb phosphorylation by cyclin D complexed with cyclin-dependent kinases (CDK) 4 and 6, which are in turn regulated by CDK inhibitors, such as p16^INK4 protein[12]. pRb is underphosphorylated throughout G₁ phase and phosphorylated just before S phase[12,13]. Hypophosphorylated pRb arrests cells in G₁ phase, and phosphorylation relieves this inhibition resulting in S phase entry[12-14]. p16^INK4 is associated with the cyclin D-CDK4 complex, preventing pRb phosphorylation and consequently, S phase entry[15-18]. Dysregulation of the p16^INK4/pRb/cyclin D1 pathway has been reported in numerous tumor types[19,20]. Inactivation of tumor suppressor gene products pRb and p16^INK4 protein is a common event in human cancers[9].

P16 gene located on chromosome 9p21, is a new tumor suppressor gene, which was identified by an American molecular geneticist in 1995 and is also called multiple tumor suppressor 1 (MTS1) for its suppressing function on multiple tumors[16]. Rb gene is the first tumor suppressor gene located at chromosome 13q14 identified by the location cloning method. The product of Rb is a nuclear phosphoprotein. Loss of pRb has been demonstrated in a variety of cancers, including gastric, pancreatic and bladder cancers, small cell lung and colorectal carcinomas[21-25].

Our study showed that the positive expression rates of p16 and Rb in gallbladder carcinoma were significantly lower than those in adenoma and cholecystitis. The positive expression rates of p16 and Rb in Nevin stagings S₁-S₂ and S₃ of gallbladder carcinoma were significantly higher than those in S₄-S₅. There was also a significant difference in p16 and Rb expressions between pathologic grades G₁ and G₃ of gallbladder carcinoma. These results suggest that loss and mutation of p16 play an important role in gallbladder carcinoma progression, and there is a consanguineous relation between p16 and invasion and metastasis of gallbladder carcinoma. p16 gene can inhibit the development of gallbladder carcinoma.

The overexpression of cyclin D1 has been reported in a wide range of human cancers[26-28]. In our study the abnormal cyclin D1 expression rates in gallbladder carcinoma and adenoma were significantly higher than those in chronic cholecystitis. No significant difference was found in cyclin D1 expression between gallbladder carcinoma and adenoma. Similarly, there was no marked difference among the pathologic grades G1, G2 and G3 and among Nevin stagings S₁-S₂, S₃ and S₄-S₅ of gallbladder carcinoma. Hui et al[29] have suggested that cyclin D1 overexpression is an early event in gallbladder carcinogenesis and could independently predict the prognosis for patients with resectable gallbladder carcinoma.

Among the 20 cases of p16-positive gallbladder cancer, 8 were Rb positive. Of 24 cases of Rb-positive gallbladder cancer, 8 were p16 positive. Tumor suppressor gene p16 is correlated with Rb. Therefore, inactivation of pRb may stimulate cells to increase p16^INK4 expression, which inhibits the activity of CDK4 and inactivates cyclin D1-CDK4 complex. On the contrary, pRb overexpression stimulates cells to increase p16^INK4 protein loss, which enhances the activity of CDK4, thus inactivating excrescent pRb.

There is a negative correlation between p16 and cyclin D1. A close association of cyclin D1 overexpression with p16^INK4 protein loss has been found in bladder cancer[30] and hepatocellular carcinoma[31,32]. Negative expression of p16^INK4 protein and positive expression of cyclin D1 protein are significantly correlated to the high invasion and metastasis of tumor and the poor survival of patients.

In conclusion, disruption of the cyclin D1/p16^INK4-pRb pathway plays an important role in the progression of gallbladder carcinoma. Loss or decreased expression of p16 and pRb has an obvious correlation with malignant degree and metastasis of gallbladder carcinoma. Cyclin D1 overexpression is an early event in gallbladder carcinogenesis.