EFFECTS OF p16^(INK4) GENE ON CHEMOSENSITIVITY OF HUMAN GLIOMA U251 CELL LINE TO TENIPOSIDE

Objective: To determine the effects on the cell growth, tumorigenicity and chemosensitivity of p16/CDK4I in human glioma. Methods: p16 gene was transfected into U251 cells by lipofectin. Expression of exogenous p16 gene was confirmed by immunohistochemistry and Northern blot. The effects of exogenous p16 gene on the growth and chemosensitivity to teniposide were examined. Results: Expression of exogenous p16 gene inhibited the growth dramatically in vitro. G1 arrest of tumor cells was observed. However, wt p16-positive U251 was less sensitive than control cell lines and the number of apoptotic cells after chemotherapy was reduced. Conclusion: The expression of exogenous p16 gene could inhibit the growth of glioma. On the other hand, the chemosensitivity to teniposide of p16-positive U251 was decreased.

Exogenous p16 gene therapy combined with X-ray irradiation suppresses the growth of human glioma cells

In this study, we infected human glioma U251 cells with a replication-defective recombinant adenovirus carrying the p16 gene. This adenovirus constructed was able to transfect exogenous p16 into the human glioma cells efficiently, and direct a high level of p16 protein expression. Tumor-inhibition experiments demonstrated that treatment with the adenovirus-p16 significantly inhibited the growth of glioma cells in vitro as well as the in vivo development of tumors in nude mice bearing a brain glioma. The combination of adenovirus-p16 gene treatment and X-ray irradiation resulted in a greater inhibition of tumor growth. Adenovirus-mediated p16 gene therapy conferred a significant antitumor effect against human glioma cells both in vitro and in vivo, and that there was a synergistic effect when X-ray irradiation was also used.

Hypermethylation of promoter 5'CpG island of p16 gene in glioma tissue and plasma

Objective: To explore the clinical significance of methylation status of promoter CpG island of p16 gene in glioma tissue and plasma. Methods: Methylation specific polymerase chain reaction (MSP) was used to determine the methylation status of the promoter for p16 gene within glioma tissue and plasma. Immunohistochemical method (SP) was used to analyze the expressions of p16 and Ki-67 proteins. Results: Hypermethylation was found in 17/40 (42.5%) of brain gliomas, in com-parison with 11/40 (27.5%) plasma specimens (χ2 = 1.9780, P = 0.1596). Loss of p16 expression was associated (P = 0.0229) with hypermethylation of CpG island of promoter regions. Hypermethylation of p16 gene CpG island was significantly related to the increase of malignant grade of brain glioma (Tissue: χ2 = 11.4288, P = 0.0007; Plasma: χ2 = 8.9439, P = 0.0028). The Ki-67 index increased significantly (P < 0.05) in brain gliomas methylated in contrast to those unmethylated. Conclusion: P16 hypermethylation may be one of the major mechanisms of tumorigenesis of gliomas. Methylated tumor-specific DNA may be as a plasma biomarker for prognosis in patients with glioma.

Polymorphism of p16INK4a gene and rare mutation of p15INK4b gene exon2 in primary hepatocarcinoma

INTRODUCTION Hepatocellular carcinoma(HCC)is the mostcommon cause of death from cancer in China.Themechanisms of hepatocarcinogenesis are not yetknown clearly,p16INK4a gene,the multiple tumorsuppressor gene 1(MTS1),encodes P16 protein,which acts as an inhibitor by binding directly toCDK4 and CDK6 and preventing its association

Tumor suppressor gene p16 and Rb expression in gastric cardia precancerouslesions from subjects at a high incidence area in northern China

AIM:To further understand the molecular basis for gastric cardia carcinogenesis and to provide etiological clues. METHODS: Endoscopic mucosa biopsy and histopathological examinations were made on 37 subjects from a high incidence area for both esophageal and gastric cardia carcinomas in northern China. All the biopsy samples were fixed in 850 ml. (-1)L alcohol and embedded in paraffin. Each block contained one piece of tissue and was serially section at 5 microm. Immunohistochemistry (ABC) was carried out on these gastric cardia samples to determine the alterations of p16 and Rb. RESULTS: Based on the histopathlogical examination there were 11 cases of chronic superficial gastritis, 12 cases of chronic atrophic gastritis and 14 cases of dysplasia. The immunostaining demonstrated different levels of unclear immunostaining of p16 and Rb in normal gastric cardia tissue and the tissues with different severity of lesions. With the lesions progressing, the positive immunostaining rates for p16 protein had a decreasing tendency. In contrast, the positive immunostaining rate for Rb protein had an increasing tendency. There was a significant negative relationship between the two parameters. Changes of p16 was CSG 11(100%), CAG 7(58%), DYS 4(29%) and changes of Rb was CSG 2(18%), CAG 8(67%) and DYS 12(86%), (P【0.05). CONCLUSION: The alterations of p16 and Rb protein may play a role in the early stages of gastric cardia carcinogenesis.

Expression of p16 gene and Rb protein in gastric carcinoma and their clinicopathological significance

AIM:To analyze the correlation between the protein expression of p16 and Rb genes in gastric carcinoma (GC), to investigate the role of p16 gene in invasion and lymph node metastasis of GC, and to examine the deletion and mutation in exon 2 of p16 gene in GC. METHODS: The protein expression of p16 and Rb genes was examined by streptavidin-peroxidase conjugated method (S-P) in normal gastric mucosa, dysplastic gastric mucosa and GC. The deletion and mutation of p16 gene were examined by polymerase chain reaction (PCR) and polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) respectively in normal gastric mucosa and GC. RESULTS: The positive rates of P16 and Rb protein expression respectively were 96% (77/80) and 99% (79/80) in normal gastric mucosa, 92% (45/50) and 80% (40/50) in dysplastic gastric mucosa, 48% (58/122) and 60% (73/122) in GC. The positive rates of P16 and Rb protein expression in GC were significantly lower than that in normal gastric mucosa and dysplastic gastric mucosa (P<0.05). The positive rate of P16 protein expression in mucoid carcinoma (10%, 1/10) was significantly lower than that in poorly differentiated carcinoma (51%, 21/41), undifferentiated carcinoma (58%, 15/26) and signet ring cell carcinoma (62%, 10/16) (P<0.05). The positive rates of P16 protein in 30 cases of paired primary and lymph node metastatic GC were 47% (14/30) and 17% (5/30) respectively, being significantly lower in the later than in the former (P<0.05). There was no mutation in exon 2 of p16 gene in the 25 freshly resected primary GCs. But five cases in the 25 freshly resected primary GCs displayed deletion in exon 2 of p16 gene. The positive rate of both P16 and Rb proteins was 16% (14/90), and the negative rate of both P16 and Rb proteins was 8% (7/90) in 90 GCs. The rate of positive P16 protein with negative Rb protein was 33% (30/90). The rate of negative P16 protein with positive Rb protein was 43% (39/90). There was reverse correlation between P16 and Rb expression in 90 GCs CONCLUSION: The loss protein expression of p16 and Rb genes is related to GC. The loss expression of P16 protein is related to the histopathologic subtypes and lymph node metastasis of GC. Expression of P16 and Rb proteins in GC is reversely correlated. The deletion but not mutation in exon 2 of p16 gene may be involved in GC.

p16 gene methylation in colorectal cancers associated with Duke's staging

AIM: To explore the association of methylation of the CpG island in the promotor of the p16 tumor suppressor gene with the clinicopathological characteristics of the colorectal cancers. METHODS: Methylation-specific PCR (MSP) was used to detect p16 methylation of 62 sporadic colorectal cancer specimens. RESULTS: p16 methylation was detected in 42% of the tumors.Dukes'staging was associated with p16 methylation status.p16 methylation occurred more frequently in Dukes'C and D patients (75.9%) than in Dukes'A and B patients (12.1%). CONCLUSION: p16 methylation plays a role in the carcinogenesis of a subset of colorectal cancer, and it might be linked to poor prognosis.

Alterations of FHIT Gene and P16 Gene in Nickel Transformed Human Bronchial Epithelial Cells

Objective To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immortal human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis. Methods 16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting. Results NiS-treated cells exhibited overlapping growth. Compared wkh that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases （P〈0.01） and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in pl6 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8. Conclusions The FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens.

DELETIONS AND POINT MUTATIONS OF p16,p15 GENE IN PRIMARY TUMORS AND TUMOR CELL LINES

Aberrations of chromosome 9 p21 22 are involved in the genesis of many forms of cancer.The gene p16 and p15 have been assigned to this region.Both p16 and p15 are an inhibitor of cyclin D cdk4,cyclin D cdk6 complex and have been implicated in a wide variety of cancer types,including the germline of patients with familial melanoma.In order to investigate and compare the status of p16,p15 gene in primary tumors and cell lines,we examined 357 primary tumors and 29 cell lines derived from diverse tumor types.In addition to analysis of these primary tumors and cell lines,blood specimens from 91 patients either with sporadic multiple cancers or from cancer prone families were also analyzed.The data showed the following:1)Homozygous deletions of p16,p15 were comparatively rare and far less common than previously reported,although hemizygous deletions were observed in a significant fraction of many tumor types;2)the incidence of p16,p15 deletions(either homozygous deletions or heterozygous deletions)varied significantly among different tumor types;3)most deletions involved in both p16 and p15 genes;4)sequence variations in the coding sequence of p16,p15 were comparatively rare among these tumor types,though mutations and polymorphisms were identified;5)some tumors which showed LOH at 9p,containing p16 and p15 gene,did not show deletions or point mutations in the p16,p15 gene.6)In a subset of retinoblastoma and osteosarcoma where no Rb gene mutations were present a significant fraction was found to contain p16,p15 gene deletions.

Effect of 5-Aza-2'-deoxycytidine on the P16 tumor suppressor gene in hepatocellular carcinoma cell line HepG2

INTRODUCTIONHepatocellular carcinoma (HCC) is one of the mostcommon human malignancies worldwide[1,2], and isclosely associated with infection of HBV and HCVand contamination of aflatoxin B1[3-6]. Althoughthe molecular mechanisms of hepatocarcinogenesisremain poorly understood, an increasing number ofgenetic abnormalities have been recognized[7-10],for example, the p16 gene[11,12] the p53gene[13-18], the E-cadherin gene[19], and the c-mycgene[20].

The effects of CYP1A1 gene polymorphism and p16 gene methylation on the risk of lung cancer in a Chinese population

Objective： To investigate the relationship between the genetic polymorphism of CYP1A1 and the genetic susceptibility to lung cancer as well as to study the effects of the methyiation in p16 gene on the risk of lung cancer in a Chinese population. Methods： A case control study was conducted among 47 cases of lung cancer and 94 controls. The genetic polymorphism of CYP1A1 was tested with method of PCR-RFLP, and a methylation-specific PCR （MSP） was performed to detect p16 methylation. Results： It showed that there was no significant difference in frequencies of the genotypes of CYP1A1 between the two groups （P 〉 0.05）. Synergistic effects were not found between smoking and CYP1AI. Methylated p16 gene was found in 44.7% （21/47） of lung cancer tissues and in 17.0% （8/47） of normal lung tissues with significant difference （P 〈 0.05）. Conclusion： The genetic polymorphism of CYP1A1 does not increase the risk of lung cancer in a Chinese population. The methylation in p16 gene may be the most common mechanism to inactivate p16 gene in lung cancer, and is not significantly associated with genotype of CYP1A1,

Effects of Exogenous p16^(ink4a) Gene on Biological Behaviors of Human Lung Cancer Cells

The effects of exogenous p16^ink4a gene on biological behaviors of human lung cancer cell line with homozygous deletion of p16^ink4a gene were investigated. Exogenous p16^ink4a gene was transfected by lipofectin into human lung cell line A549, in which p16^ink4a gene was homozygously deleted. The expression of p16^ink4a mRNA and protein was detected by RT-PCR and immunocytochemistry, respectively. The changes in the behaviors of the transfected cell lines in vitro and in vivo were observed. In the transfected cell line A549, the exogenous p16^ink4a gene could be stably expressed. The growth of A549 cells transfected with p16^ink4a gene was obviously slowed down. Flow cytometry revealed that transfection of the exogenous p16^ink4a gene resulted in A549 cell lines arrest in G1 phase of cell cycle. The tumorigenicity of these transfected cells in nude mice could be inhibited, and the tumor growth of nude mice was significantly suppressed. It was concluded that exogenous p16^ink4a gene may be stably expressed in human lung cancer cell line A549. The expression of the introduced p16^ink4a could block lung cancer cells to entry into S phase of cell cycle and inhibit tumor malignant growth both in vitro and in vivo.

The Effects of Wild-type p5 3Gene Transfection on the Growth and Chemotherapeutic Sensitivity of Human Glioma Cells

To evaluate the effects of wild type p53 gene on the growth and chemotherapeutic sensitivity of human glioma cells, plasmid PC53 SN3 carrying wild type p53 gene was transfected into U251 cells. p53 gene expression in transfected cells was detected by RT PCR, the cell growth inhibition and apoptosis in either the absence or the presence of cisplatin was assessed by MTT and flow cytometry. The transfection of p53 gene into U251 cells was confirmed by RT PCR. MTT showed that p53 gene by itself induced strong inhibition effect on the growth of U251 cells [inhibition rate, IR (79.60±5.69) %]. The killing effects of cisplatin by itself on U251 cells was not strong [IR (19.40±6.69) %, (24.41±2.68) %, (51.84±13.38) %, (66.22±5.02) %] and increased with the increase of cisplatin concentration (1, 2, 4, 8 μg/ml). When combined treatment of wild type p53 gene transfection and cisplatin was used, that was significantly increased [IR (91.64±1.00) %, (94.98±1.67) %, (95.32±2.01)%, (95.65±1.00) %]. The apoptosis rate of U251 cells induced by p53 gene transfection was 17.38%. That induced by cisplatin increased (5.71 %, 5.93 %, 6.27 %, and 6.81 %) with the increase of cisplatin concentration (1, 2, 4, 8 μg/ml). The apoptosis rate was also significantly increased (23.50 %, 23.54 %, 23.89 %, and 28.88 %) after combined treatment of p53 and cisplatin with different concentration (1, 2, 4, 8 μg/ml). It is concluded that wild type p53 gene and cisplatin could result in synergistic inhibition effects on the growth of human glioma cells.

PROMOTER HYPERMETHYLATION OF p16 GENE AND DAPK GENE IN SERA FROM HEPATOCELLULAR CARCINOMA (HCC) PATIENTS

Objective： To analyze the aberrant methylation of p16 gene and DAPK gene in sera from primary liver cancer patients ad to evaluate the clinical significance. Methods： A methylation-specific PCR was performed for the detection of promoter hypermethylation of p16 gene and DAPK gene in blood DNA from 64 cases of HCC patients, and to analyze the relation of the aberrant methylation of p16 gene and KAPK gene and the clinical pathological data. Results： 76.6%（49/64） of the sera from 64 cases of HCC patients showed hypermethylation for p16 promoter and 40.6% （26/64） for KAPK promoter, whereas no methylated p16 gene promoter and DAPK gene promoter were found in sera from benign liver diseases patients and normal control. Methylated p16 gene and KAPK gene promoters in sera did not strongly correlated with HBsAg, stage, metastasis and differentiation in HCC; but strongly correlated with AFP. Conclusion： Detection of the aberrant methylation of p16 gene and KAPK gene in blood DNA from HCC patients might offer an effective means for the earlier auxiliary diagnosis of the malignancy.

Quantitative Study on Expression of P16 Multiple Tumor Suppressor Gene in Salivary Gland Neoplasm

The expression of P16 gene were found in all 3 groups. The positive unit (PU) was higher in tumor group and cancer group than that in normal group ( P <0.01). Furthermore, the PU of P16 was stronger in cytoplasm than in nucleus. Malignant tumors and acini surrounding the tumor revealed strong positives and week positives respectively. The PU of P16 gene was higher in deep lobe of recurrent parotid neoplasm with incomplete capsule than that in shallow lobe of primary parotid neoplasm with complete capsule. Our findings suggests that P16 gene plays equally important role in the salivary gland tumors and tumors in other part of the body.

Effect of Wild-type p53 Gene Transfection on the Growth and Radiotherapeutic Sensitivity of Human Glioma Cells

Summary： To evaluate the effect of wild type p53 gene on the growth and radiotherapeutic sensitivity of human glioma cells, plasmid PC53 SN3 carrying wild type p53 gene was transfected into U251 cells, p53 gene expression in transfected cells was detected by RT PCR, and the cell growth inhibition and apoptosis in the absence or presence of irradiation were assessed by MTT and flow cytometry. The transfection of p53 gene into U251 cells was confirmed by RT PCR. MTT showed that p53 gene alone induced strong inhibitory effect on the growth of U251 cells （inhibition rate （IR）： （79.60±5.69）%）, The killing effect of irradiation alone on U251 cells was not strong （IR： （17. 06±4.35）%, （17.39±1.67）%, （18.73±4.68）%） and increased with the irradiation doses （3, 6, 9 Gy）. When combined treatment of wild-type p53 gene transfection and irradiation was used, the effect was significantly increased （IR：（80.60±5.35）%, （90.30＋1.67） %, （91.30±2.01） %）. Theapoptosis rate of U251 cells induced by p53 gene transfectionwas 17.38 %. The rate induced by irradiation increased （4. 61%, 4. 84%, 5. 40 %） with the irradiation doses （3, 6, 9 Gy）. Theapoptosis rate was also significantly increased （17.80%, 20.03%, 22.34%） after combined treatment of p53 and irradiation with different doses （3, 6, 9 Gy）. It is concluded that wildtype p53 gene and irradiation could result in synergistic inhibitory effect on the growth of human glioma cells.

METHYLATION OF p16 AND p15 GENES IN MULTIPLE MYELOMA

Objective.To investigate the frequency of p16a nd p15gene methylation in multiple myeloma (MM),and its relationship with bone marrow ce ll apoptosis and clinical outcome.Methods.Twenty-two patients with MM were stu died to detect p16and p15gene methylation.Methyla-tion-specific polymerase chain rea ction(MSP)was used to detect gene methylation,and terminal trans-ferase-mediated dUTP nick end-labeling(TUNEL)was used to detect cell apoptosis.Results.p16and /or p15gene methylatoin was d etected in 10of 22patients(45.4%).There were 3pa-tients with p16gene methylation,9p atients with p15gene methylation,a nd 2patients with both genes methyla-tion.The incidence of methylation o f p15gene was higher than that of p16g ene(P<0.05).The patients with p16and /or p15gene methylation had a delayed cell apoptosis,poor respon se to chemotherapy,and a short over-all survival(OS).Conclusion.The methylation of p16and /or p15gen e plays a key role in MM apoptosis path ogenesis.The patients with both p16and p15gene me thylation had a poor prognosis.

STUDY OF DELETION OF P16 GENE IN THE PROGRESSION OF BRAIN ASTROCYTOMAS

Objective: To study the relationship between deletion of P16 gene and occurrence and progression of astrocytomas Methods: The techniques of polymerase chain reaction (PCR) and immunohistochemistry were used to detect the deletion of exon2 of P16 gene and expression of P16 gene in 52 cases of Brain astrocytoma Results: The deletion rate of exon2 of P16 gene in the tumors analyzed was 34 6% Most of them with deletion of exon2 of p16 gene were high grade astrocytomas (grade III 42%, grade IV 50%) 61 5% of the tumors were absent from expression of p16 and the deletion rate of p16 protein increased with the grade of astrocytoma (X 2=10 83, P <0 005) Conclusion: Deletion of p16 gene and protein may correlate with the malignant progression of astrocytoma

Gene Product Expression of Cyclin D_2 and p16 During the Transition from Cardiac Myocyte Hyperplasia to Hypertrophy

The current study was to investigate mRNA expression of cyclin D 2 and p16 during the transition from cardiac myocyte hyperplasia to hypertrophy. Cultured cardiac myocytes (CM) and fibroblasts (FC) obtained from 1 day old Sparague Dawley rats were used in this study. We have determined (1) hyperplasia by cell growth curve and fluorescence activated cell sorting (FACS); and (2) ultrastructure by electron microscope observation; and (3) expressions of cyclin D 2 mRNA and p16 mRNA by using in situ hybridization and image analysis. The results were shown (1) Results of cell growth curve and FACS analysis showed CM could proliferate in the first 3 cultured days (4 days in postnatal development). But the ability decreased quickly, concomitant with the differentiation. (2) The ultrastructure of CM showed the large amount of myofilaments and mitochondrion and FC showed moderate amount of rough endoplasmic reticulum. (3) The expression of cyclin D 2 mRNA in 3 , 4 , 5 day CM group was 0.89 times(p<0.05), 0.80 times (p<0.05)and 0.56 times (p<0.01)of that in 1 day group respectively. P16 mRNA in 2 , 3 , 4 , 5 day CM group were 1.63 times(p<0.01),1.72 times(p<0.01),1.99 times (p<0.01)and 2.84 times (p<0.01) of that in 1 day group respectively. It can be concluded that cultured neonatal rat cardiac myocytes could proliferate during the first 3 cultured days, but the ability of proliferation decreased, from the fourth day, concomitant with differentiation. Cyclin D 2 and p16 have the key roles during the transition from myocyte hyperplasia to hypertrophy.