1.The alteration of MTS1 gene in precancerous lesions and squamous cell carcinoma of oral mucosa.
Wen-xia WANG ; Shan-zhen SUN ; Pi-shan YANG ; Bo-long MA ; Yan LONG
Chinese Journal of Stomatology 2003;38(5):361-363
OBJECTIVETo investigate the expression and alteration (including homozygous deletion and mutation) of MTS1 gene in precancerous lesions and squamous cell carcinomas (SCC) of oral mucosa, and to analyse the function of MTS1 gene alteration in oral mucosal carcinogenesis.
METHODSThe expression of p16 protein produced by MTS1 gene was examined with immunohistochemical SP method in 10 normal oral mucosas, 30 precancerous lesions (10 mild, 10 moderate and 10 severe dysplasia respectively) and 45 squamous cell carcinomas (SCCI18, SCCII 19, SCCIII 8). The deletion and mutation of exon1 and exon2 of MTS1 gene were examined with methods of PCR and SSCP in these same samples.
RESULTSAll the precancerous lesions had p16 protein expression and no alteration of MTS1 gene. In SCC, the positive rate of p16 protein was 60.0% with 72.2% in SCCI, 57.9% in SCCII, 37.5% in SCC III, and there were no significant difference among the three groups by chi2 test (P>0.05). Gene homozygous deletion of exon1 and/or exon2 was detected in 10 cases, and gene mutation in 4 cases. The whole rate of gene alteration was 31.1% (14/45). The MTS1 gene alteration rate was 27.8% in SCCI, 31.6% in SCCII, 37.5% in SCC III and there was also no significant difference among the three groups by chi2 test (P>0.05). In SCC with local lymph nodes metastasis, MTS1 alteration rate was 57.1%, while in SCC with no lymph nodes metastasis was 8.3%, and there was significant difference by chi2 test (P<0.05).
CONCLUSIONSMTS1 gene alteration is not an early event in the carcinogenesis of oral mucosa and can not be used as a biology mark to examine oral precancerous lesions. MTS1 gene may play a certain role in the progression of oral squamous cell carcinomas.
Carcinoma, Squamous Cell ; chemistry ; genetics ; pathology ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Genes, p16 ; Humans ; Lymphatic Metastasis ; Mouth Neoplasms ; chemistry ; genetics ; pathology ; Mutation ; Precancerous Conditions ; genetics
2.Analysis on point mutation of the CDKN2/p16 gene in lung cancer.
Changqing SU ; Yukun YE ; Dong WANG ; Xiangrong CAO ; Shufeng LI ; Xiangnian SHAN
Chinese Journal of Medical Genetics 2002;19(1):37-40
OBJECTIVETo study the status of CDKN2/p16 gene point mutation in lung cancer.
METHODSPolymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing were used to detect the point mutation of CDKN2/p16 gene exon 2 in 89 cases of lung cancer.
RESULTSIn 69 cases of the lung cancer without deletion of CDKN2/p16 gene exon 2, 16 cases were found to have suspicious abnormality of CDKN2/p16 gene exon 2 by PCR-SSCP, and in these 16 cases, 9 were found to harbor point mutations of CDKN2/p16 gene exon 2 by automated sequencing analysis.
CONCLUSIONThe point mutation is one of the mechanisms for CDKN2/p16 gene inactivation, but it is not the chief mechanism. The inactivation of CDKN2/p16 gene aroused by point mutation plays a role to some extent in the genesis and progression of lung cancer.
Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Exons ; Humans ; Lung Neoplasms ; genetics ; Point Mutation ; Polymerase Chain Reaction ; methods ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis, DNA ; methods
3.Effects of hexamethylene bisacetamide on cell cycle and expression of its regulatory proteins in HL-60 cells.
Qin-Hong WANG ; Yi XIE ; Hua-Hua FAN ; Li GAO ; Yan LIU
Journal of Experimental Hematology 2003;11(5):480-484
Hexamethylene bisacetamide (HMBA) is referred as a differentiation-inducer for the clinical treatment of acute myeloid leukemia and myelodysplastic syndrome. However, the molecular mechanism of the effects of HMBA on myeloid leukemic cells remains unknown. In this study, the effects of HMBA on cell cycle and expression of cell cycle regulatory proteins in HL-60 cell were investigated in order to explore its pharmacological mechanism. The altered distribution of cell cycle and expression of its regulatory proteins (cyclin D, cyclin E and p27) in HL-6 0 cell induced by HMBA were analyzed by flow cytometry. The effects on transcription for mRNA of CKI p15, p16 and p27 in HL-60 cell were further studied by RT-PCR. The results showed that HMBA could mainly commit HL-60 cell to G0/G1 arrest and the significantly decreased endocytic cyclin E protein and increased cyclin D/p27 protein after HMBA treatment were found. There was no expression of p15, p16 mRNA in untreated HL-60 cell and 3 mmol/L of HMBA could make them expressed after exposed for 24 h or 48 h respectively. The expression of p27 mRNA was positive and no obviously different in untreated HL-60 cells exposed for 24 h, 48 h and 72 h. These results suggested that one of the pharmacological mechanisms of HMBA was to elevate the expression of p27 and reduce the cyclin E expression as well as to activate the expression of p15, p16 gene mRNA, that arrested cell at G0/G1 and exerted its effects of anti-proliferation.
Acetamides
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pharmacology
;
Antineoplastic Agents
;
pharmacology
;
Cell Cycle
;
drug effects
;
Cell Cycle Proteins
;
analysis
;
genetics
;
Cyclin D
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Cyclin E
;
analysis
;
Cyclin-Dependent Kinase Inhibitor p15
;
Cyclin-Dependent Kinase Inhibitor p27
;
Cyclins
;
analysis
;
Genes, p16
;
HL-60 Cells
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Humans
;
RNA, Messenger
;
analysis
;
Tumor Suppressor Proteins
;
analysis
;
genetics
4.Relationship between alterations of p16INK4a and p14ARF genes of CDKN2A locus and gastric carcinogenesis.
Shao-Hui TANG ; Dong-Hua YANG ; He-Sheng LUO ; Jie-Ping YU ; Jian-Chang SHU
Chinese Journal of Epidemiology 2004;25(6):517-521
OBJECTIVETo investigate the relationship between alterations of p16INK4a and p14ARF genes and gastric carcinogenesis.
METHODSTumors and gastric tissues neighboring carcinoma from 48 patients with gastric cancer were studied. Homozygous deletion, mutation, methylation of the CpG islands, mRNA expression of p16INK4a and p14ARF genes were assessed by polymerase chain reaction (PCR), PCR-single strand conformation polymorphism (SSCP), PCR based methylation assay, and reverse transcription (RT)-PCR.
RESULTS(1) The overall homozygous deletion rate of p16INK4a and p14ARF was 35.4% (17/48), and no homozygous deletion was examined in all the gastric tissues neighboring tumor. (2) There was no point mutation of p16INK4a and p14ARF in 31 gastric cancers without homozygous deletion and in the matched gastric tissues adjacent to tumor. (3) Methylation of the CpG islands of p16INK4a and p14ARF was detected in 47.9% (23/48) of gastric cancers, while methylation was observed only in 2 of 48 gastric tissues neighboring cancers with a significant difference (P <0.01). (4) The rate of p16INK4a mRNA loss was 47.9% (23/48) in gastric cancer, and the cases of combined methylation of exons 1alpha and 2 had a higher loss rate (100%, 6/6) of p16INK4a mRNA than those of methylation form the other exons (11.8%, 2/17) (P <0.01). The loss rate of p14ARF mRNA was 45.8% (22/48) in gastric cancer, and patients with combined methylation of exons 1beta and 2 had a higher loss rate (100%, 3/3) of p14ARF mRNA than those of the methylation from the other exons (15%, 3/20) (P < 0.05). (5) The combined loss of p16INK4a and p14ARF mRNAs was examined in 1 (5.6%) of 18 cases of well and moderately-differentiated carcinomas, and 11 (36.7%) of 30 cases of poorly and not-differentiated carcinomas with significant difference (P <0.05).
CONCLUSIONp16INK4a and p14ARF genes were frequently inactivated by homozygous deletion and methylation of the 5' CpG islands in gastric cancer, which might have played an important role in the development of gastric cancer.
Adenocarcinoma ; genetics ; Adolescent ; Adult ; Aged ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA Methylation ; Female ; Gene Deletion ; Genes, p16 ; Humans ; Male ; Middle Aged ; RNA, Messenger ; analysis ; Stomach Neoplasms ; genetics ; Tumor Suppressor Protein p14ARF ; genetics
5.Relationship between alterations of p16(INK4a) and p14(ARF) genes of CDKN2A locus and gastric carcinogenesis.
Shaohui TANG ; Hesheng LUO ; Jieping YU ; Donghua YANG ; Jianchang SHU
Chinese Medical Journal 2003;116(7):1083-1087
OBJECTIVETo investigate the relationship between alterations of p16(INK4a) and p14(ARF) genes and gastric carcinogenesis.
METHODSThe tumors and neighboring gastric tissues from 48 patients with gastric cancer were studied. The homozygous deletion, mutation, methylation of the CpG islands, and mRNA expression of p16(INK4a) and p14(ARF) genes were assessed by PCR, PCR-SSCP, PCR based methylation assay, and RT-PCR.
RESULTS(1) The homozygous deletion rate of p16(INK4a) and p14(ARF) was 35.4% (17/48), and no homozygous deletion was examined in any gastric tissue neighboring the tumor. (2) There was no point mutation of p16(INK4a) and p14(ARF) in 31 gastric cancers without homozygous deletion or in the matched gastric tissues adjacent to the tumor. (3) Methylation of the CpG islands of p16(INK4a) and p14(ARF) was detected in 47.9% (23/48) of gastric cancers, while methylation was observed only in 2 of 48 gastric tissues neighboring the cancer with a significant difference (P < 0.01). (4) The loss rate of p16(INK4a) mRNA was 47.9% (23/48) in gastric cancer, and the patients of the combined methylation of exons 1alpha and 2 had a higher loss rate (100%, 6/6) of p16(INK4a) mRNA than those of the methylation of the other exons (11.8%, 2/17, P < 0.01); the loss rate of p14(ARF) mRNA was 45.8% (22/48) in gastric cancer, and patients with the combined methylation of exons 1beta and 2 had a higher loss rate (100%, 3/3) of p14(ARF) mRNA than those of the methylation of the other exons (15%, 3/20, P < 0.05). (5) The combined loss of p16(INK4a) and p14(ARF) mRNAs was examined in 1 (5.6%) of 18 patients of well and moderately-differentiated carcinomas, and 11 (36.7%) of 30 patients of poorly and not-differentiated carcinomas with a significant difference (P < 0.05).
CONCLUSIONp16(INK4a) and p14(ARF) genes are frequently inactivated by homozygous deletion and methylation of the 5'CpG islands in gastric cancer, which may play an important role in the carcinogenesis of gastric cancer.
Adolescent ; Adult ; Aged ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA Methylation ; Gene Deletion ; Genes, p16 ; Humans ; Middle Aged ; RNA, Messenger ; analysis ; Stomach Neoplasms ; genetics ; Tumor Suppressor Protein p14ARF ; genetics
6.Role of HPV DNA detection and p16(INK4A) protein expression in diagnosis of endocervical adenocarcinoma.
Zhi-Lan MENG ; Li-Na GUO ; Yu-Feng LUO ; Jin-Ling CAO ; Jian-Wei WAN ; Tong-Hua LIU
Chinese Journal of Pathology 2007;36(12):810-813
OBJECTIVESTo evaluate the significance of p16(INK4A) protein expression and positivity for HPV DNA in distinguishing between endocervical and endometrial adenocarcinoma.
METHODSExpression of p16(INK4A) protein in 30 cases of endocervical adenocarcinoma and 10 cases of endometrial adenocarcinoma was assessed by immunohistochemistry. In-situ hybridization for human papillomavirus (HPV) DNA was also performed in 20 cases of endocervical adenocarcinoma and 10 cases of endometrial adenocarcinoma.
RESULTSThe positive rate for p16(INK4A) in endocervical adenocarcinoma was 70% (21/30), as compared with 30% (3/10) in endometrial adenocarcinoma. The tumor cells in endocervical adenocarcinoma showed diffuse and strong expression of p16(INK4A) protein with both cytoplasmic and nuclear staining. In contrast, the endometrial adenocarcinoma cells showed patchy and weak expression of p16(INK4A). On the other hand, HPV DNA (type 16 or 18) was detected by in-situ hybridization in 9 (45%) of the 20 cases of endocervical adenocarcinoma and none of the 10 cases of endometrial adenocarcinoma.
CONCLUSIONSThe expression of p16(INK4A) protein is significantly higher in endocervical adenocarcinoma than in endometrial adenocarcinoma. This expression pattern can serve as a useful immunohistochemical marker in the differential diagnosis. p16(INK4A) protein immunohistochemistry appears to be more sensitive than HPV DNA testing in distinguishing between endocervical and endometrial adenocarcinoma, especially in biopsy or curettage specimens.
Adenocarcinoma ; diagnosis ; genetics ; virology ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA, Viral ; analysis ; Endometrial Neoplasms ; diagnosis ; genetics ; virology ; Female ; Gene Expression Regulation, Neoplastic ; Human papillomavirus 16 ; genetics ; Human papillomavirus 18 ; genetics ; Humans ; In Situ Hybridization ; Uterine Cervical Neoplasms ; diagnosis ; genetics ; virology
7.Two isomers of HDTIC isolated from Astragali Radix decrease the expression of p16 in 2BS cells.
Pei-chang WANG ; Zong-yu ZHANG ; Jian ZHANG ; Tan-jun TONG
Chinese Medical Journal 2008;121(3):231-235
BACKGROUNDAstragali Radix, the root of Astragalus membranceus (Fish) Bunge Var. mongholicus (Bge), is a crude drug considered as one of the effective traditional Chinese anti-ageing material. The two isomers of 4-hydroxy-5-hydroxymethyl-[1, 3] dioxolan-2, 6'-spirane-5', 6', 7', 8'-tetrahydro-indolizine-3'-carbaldehyde (HDTIC), HDTIC-1 and HDTIC-2, were first extracted from the herb in 2002. We demonstrated previously that 0.1 micromol/L HDTIC-1 or 1.0 micromol/L HDTIC-2 strongly delay replicative senescence of human fetal lung diploid fibroblasts (2BS). In this study, we chose them to investigate their effects on the expression of senescence-associated genes to explore the mechanism of how HDTIC delays replicative senescence.
METHODSThe effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 were observed in vitro by RT-PCR and Western blot. The anti-oxidative activities of the compounds were also observed by phenotype alteration after treatment with antioxidants.
RESULTSThere was an obvious expression of p16 in the control senescent cells. However, in the 2BS cells, after 56 population doublings (PDs) grown from PD28 in 0.1 micromol/L HDTIC-1 or 1.0 micromol/L HDTIC-2, there was a weak mRNA expression of p16 and no protein expression of p16 was observed. The expression level of p21 increased with cell ageing. Moreover, there was no difference between the expression level of p21 in the control cells and that in the same PD cells cultured with HDTIC compounds. The results also showed that 2BS cells exposed to 100 micromol/L H2O2 for 5 minutes return to their non-senescent phenotype and continue to be confluent after incubating the damaged cells with HDTIC-1 (1.0 micromol/L ) or HDTIC-2 (10 micromol/L ) for 1 hour.
CONCLUSIONSExpression of p16 by 2BS cells was strongly inhibited by HDTIC compounds, which could contribute to their delayed replicative senescence by the way of p16(INK4a)/Rb/MAPK. The anti-oxidative activities of HDTIC-1 and HDTIC-2, described in this study for the first time, might be indirectly related to their inhibition of p16 expression.
Antioxidants ; pharmacology ; Astragalus Plant ; chemistry ; Cells, Cultured ; Cellular Senescence ; drug effects ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; analysis ; genetics ; Dioxolanes ; pharmacology ; Female ; Fibroblasts ; chemistry ; drug effects ; metabolism ; Humans ; Indolizines ; pharmacology ; Plant Roots ; chemistry ; RNA, Messenger ; analysis
8.Impact of p16INK4A and p15INK4B on human hepatocellular carcinoma cell proliferation and apoptosis.
Yang QIN ; Jian-yu LIU ; Bo LI ; Wen-zhen PENG ; Ming-de FU ; Zhi-lin SUN ; Ze-fang SUN
Chinese Journal of Medical Genetics 2004;21(2):132-137
OBJECTIVEBoth tumor suppressor p16INK4A and p15INK4B are members of INK family of CDK inhibitor. Although the role of p16 has been well documented, the role of p15 and its signaling pathway remain less well studied. This study was aimed to assess the effect of p16 and p15 on hepatocarcinoma cell lines with different status of Rb gene.
METHODSAfter identification of the genetic status of p16, p15 as well as Rb of human hepatocellular carcinoma (HCC) cell lines BEL7402, SMMC7721 with the use of multiple PCR, the eukaryotic expression p16 and p15 recombinants pXJ-p16 and pXJ-p15 were constructed, respectively. The existence of exogenous p16, p15 genes, and the expression of p16 and p15 were assayed by means of PCR and RNA dot blotting. Finally, the proliferation and apoptosis were studied by using MTT, colony formation assay and flow cytometry.
RESULTSNeither deletion of p16 nor p15 was detected in the two cell lines. However, Rb exons 14-16 instead of exons 22-23 deletion existed in SMMC7721. The increased mRNA expression level of p16 was found in BEL7402-p16 and SMMC7721-p16, while increased mRNA expression level of p15 was found in BEL7402-p15. The endogenous p16 and p15 genes were transcripted at low level. The cell growth and colony formation were decreased in BEL7402-p15, compared with either mock cell BEL7402 or vector control cell BEL7402-pXJ. Also shown in this study were an altered G1 phase population from 37.7% to 43.6%, an S phase population from 22% to 13% (P<0.05), and a Sub G1 peak (apoptosis peak) in BEL7402-p15. Conversely, BEL7402-p16 with endogenous p16 gene showed neither difference in cell cycle population nor difference in colony formation rate, compared with control cell groups. Additionally, SMMC7721-p16 cell growth was not inhibited by exogenous p16 gene.
CONCLUSIONp15 significantly arrested cell proliferation and induced apoptosis in BEL7402 in vitro, and the function was not influenced by endogenous p15 gene. The inhibition of cell growth by p16 on HCC cells could be dependent on intact RB pathway.
Apoptosis ; Carcinoma, Hepatocellular ; genetics ; Cell Cycle Proteins ; genetics ; Cell Division ; Cyclin-Dependent Kinase Inhibitor p15 ; Genes, Retinoblastoma ; Genes, Tumor Suppressor ; Genes, p16 ; Humans ; Liver Neoplasms ; genetics ; pathology ; RNA, Messenger ; analysis ; Tumor Suppressor Proteins ; genetics
9.Clinical Relevance of High-Resolution Single Nucleotide Polymorphism Array in Patients with Relapsed Acute Lymphoblastic Leukemia with Normal Karyotype: A Report of Three Cases.
Sang Hyuk PARK ; Seung Hee LEE ; Shine Young KIM ; Sun Min LEE ; Jongyoun YI ; In Suk KIM ; Hyung Hoi KIM ; Chulhun Ludgerus CHANG ; Eun Yup LEE
Annals of Laboratory Medicine 2015;35(1):132-136
We report three patients with normal karyotype (NK) ALL, who showed genetic aberrations as determined by high-resolution single nucleotide polymorphism array (SNP-A) analysis at both diagnosis and relapse. We evaluated the clinical relevance of the SNP-A assay for the detection of subtle changes in the size of affected genetic lesions at relapse as well as the prognostic value of the assay. In our patients, application of the SNP-A assay enabled sensitive detection of cryptic changes affecting clinically important genes in NK ALL. Therefore, this assay seems to be more advantageous compared to other conventional methods such as FISH assay, HemaVision (DNA Technology, Denmark), and conventional karyotyping for the detection of an "unstable genotype" at relapse, which may be associated with microscopic clonal evolution and poor prognosis. Further comprehensive studies are required to confirm the issues presented by our case patients in this report.
Adult
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Cyclin-Dependent Kinase Inhibitor p16/genetics
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Female
;
Genotype
;
Humans
;
In Situ Hybridization, Fluorescence
;
Karyotype
;
Karyotyping
;
Loss of Heterozygosity
;
Male
;
Middle Aged
;
Oligonucleotide Array Sequence Analysis
;
Polymorphism, Single Nucleotide
;
Precursor Cell Lymphoblastic Leukemia-Lymphoma/*diagnosis/genetics
;
Recurrence
;
Retinoblastoma Protein/genetics
10.Construction of antisense Bmi-1 expression plasmid and its inhibitory effect on K562 cells proliferation.
Xiu-xiang MENG ; Wei-hong LIU ; Dan-dan LIU ; Xin-yu ZHAO ; Ben-li SU
Chinese Medical Journal 2005;118(16):1346-1350
BACKGROUNDBmi-1 gene determines the proliferative capacity of normal and leukemia stem cells. Expression of Bmi-1 has been found in all types of myeloid leukemia cells in both humans and mice. This study aimed at assessing the effect of antisense Bmi-1 expression on K562 cells proliferation and p16 protein (p16) expression.
METHODSA transcriptional repressor, Bmi-1 cDNA was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) of its mRNA from K562 cells. A plasmid expressing antisense Bmi-1 mRNA was then constructed by reverse design of PCR primers and cloned to the plasmid pLNCX2; G418 was added to the medium after the plasmid was successfully introduced in K562 cells by lipofectin-mediated DNA transfection. The effects of the antisense expression on the proliferation of K562 cells were analyzed by using microculture tetrazolium and colony forming. Cell cycle was analyzed by using flow cytometry. The p16 expression of K562 cells was observed by immunofluorescence histochemical stain.
RESULTSK562 cells transfected with antisense Bmi-1 plasmid grew significantly slower than that of controls (the parental K562 and cells transfected with empty plasmid). The colony forming ability of antisense Bmi-1 plasmid transfected cells decreased significantly (P < 0.01) compared with controls. The p16 expression of cells transfected with antisense Bmi-1 was upgraded more apparently than that of controls.
CONCLUSIONThe antisense Bmi-1 gene can inhibit the growth of K562 cell and upgrade expression of p16 in K562 cells.
Cell Cycle ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Humans ; K562 Cells ; Nuclear Proteins ; antagonists & inhibitors ; genetics ; Plasmids ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; antagonists & inhibitors ; genetics ; RNA, Antisense ; physiology ; Repressor Proteins ; antagonists & inhibitors ; genetics