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 ; Cyclin-Dependent Kinase Inhibitor p16/genetics ; 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

BACKGROUND: The primary purpose of this study was to investigate the prevalence and characteristics of p16 methylation and determine the prognostic implications of the clinical data, hematologic data, and p16 methylation changes in plasma cell myeloma (PCM). METHODS: We reviewed clinical characteristics and results of laboratory tests and investigated the response to combination chemotherapy and survival time. DNA methylation of the p16 gene was tested by methylation-specific PCR. Clinical significance was evaluated. RESULTS: A total of 103 patients were enrolled in this study. The median patient age was 59.0 yr at diagnosis and the male to female ratio was 1.15:1. According to the International Staging System (ISS), patients were diagnosed as stage: I (N=17, 16.5%), II (N=41, 39.8%), III (N=39, 37.9%), or not classified (N=6). Forty-five (43.7%) patients and 36 (35.0%) patients showed abnormal karyotype and complex karyotype, respectively, on the chromosome study. The p16 methylation was observed in 39 (37.9%) of 103 patients, but there was no significant association between p16 methylation status and other clinical or laboratory factors and survival outcome. Male gender, albumin, and complex karyotype were independent prognostic factors for overall survival according to multivariate analysis (P<0.05). CONCLUSIONS: The male gender, low serum albumin level, and complex karyotype were independent poor prognostic factors for PCM. p16 methylation was relatively common in PCM, but did not influence the survival outcome.
Adult ; Aged ; Aged, 80 and over ; Antineoplastic Agents/therapeutic use ; Cyclin-Dependent Kinase Inhibitor p16/*genetics ; *DNA Methylation ; Female ; Humans ; Karyotyping ; Male ; Middle Aged ; Multiple Myeloma/drug therapy/*genetics/mortality ; Neoplasm Staging ; Polymerase Chain Reaction ; Prognosis ; Republic of Korea ; Serum Albumin/analysis ; Sex Factors ; Survival Rate

Acid Anhydride Hydrolases ; genetics ; metabolism ; Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Biomarkers, Tumor ; analysis ; Carcinoma, Squamous Cell ; diagnosis ; genetics ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; DNA Methylation ; drug effects ; Genes, p16 ; Humans ; Laryngeal Neoplasms ; diagnosis ; genetics ; metabolism ; pathology ; Neoplasm Proteins ; genetics ; metabolism ; Tumor Suppressor Proteins ; genetics ; metabolism

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

OBJECTIVEIn cervical lesions, the overexpression of p16INK4a has been reported to be closely associated with human papillomavirus (HPV) infection. This study is designed to evaluate the role of p16INK4a as a biomarker in liquid-based cervical cytology screening.

METHODSSeventy-four specimens from the patients in our hospital were collected for this study. After cytological examination with liquid-based cervical smears, high-risk HPV (HR-HPV) DNA was then detected by Hybrid Capture II assay, and the rest cells were immunostained for p16INK4a.

RESULTSOf the 74 specimens, 10 were diagnosed as negative, 15 as atypical squmous cells of undetermined significance (ASC-US), 28 as low-grade squamous intraepithelial lesion (LSIL), 5 as atypical squmous cells which could not be excluded as HSIL (ASC-H), 11 high-grade squamous intraepithelial lesion (HSIL) and 5 as squamous cell carcinoma (SCC). The positive specimens of p16INK4a were 2, 5, 8, 3, 9, 5, respectively, in the above subgroups; meanwhile, the positive specimens of HR-HPV were 1, 4, 9, 3, 7, 5, respectively, in the above groups. The positive rate of both p16INK4a and HR-HPV in HSIL, ASC-H and SCC were obviously higher than that in LSIL, ASC-US and negative cases.

CONCLUSIONPositive rate of p16INK4a and HR-HPV is highly correlated with the grade of the cervical lesion. p16INK4a immunocytochemical staining may be used as a biomarker to increase the sensitivity of cervical cytology screening and the specificity of HPV test.

Adult ; Aged ; Biomarkers, Tumor ; analysis ; Carcinoma, Squamous Cell ; chemistry ; diagnosis ; virology ; Cervical Intraepithelial Neoplasia ; chemistry ; diagnosis ; virology ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Cytodiagnosis ; methods ; DNA, Viral ; analysis ; Female ; Humans ; Immunohistochemistry ; Middle Aged ; Papillomaviridae ; genetics ; Papillomavirus Infections ; diagnosis ; metabolism ; Uterine Cervical Dysplasia ; chemistry ; diagnosis ; virology ; Uterine Cervical Neoplasms ; chemistry ; diagnosis ; virology ; Vaginal Smears ; methods ; Young Adult

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

OBJECTIVETo evaluate the value of p16INK4a immuncytochemical examination in cytological screening of cervical carcinoma and precancerous lesions.

METHODSp16JNK4a immuncytochemical detection was performed on 220 specimens remaining from liquid-based cytology, followed up by biopsy histology , and compared with the results of high-risk human papillomavirus ( HR - HPV ) DNA tests . Results In patients with cytological diagnosis of squamous cell carcinoma( SCC) , high-grade squamous intraepithelial lesion (HSIL) , low-grade squamous intraepithelial lesion (LSIL) , atypical squamous cells-cannot exclude HSIL (ASC-H) , and atypical squamous cells of undetermined significance (ASC-US) , the positive rates of p16INK4a were 100.0% (7/7), 92. 2% (107/116) , 24. 3% (17/70) , 100. 0% (14/14) and 36.4% (4/ 11) , respectively. In 111 of the 150 p6INK4a positive cases, we found 97 (87.4% ) cases which had biopsy diagnosises of > or =CIN2, but none in 18 of 70 p16INK4a negative cases was. The difference in the positive rates for p16INK4a between cervical intraepithelial neoplasia (CIN) 1 and > or =CIN2 lesions had statistical significance (P < 0. 01) , whereas for HR-HPV DNA test it was not.

CONCLUSIONp16LNK4a is over-expressed in a HSIL, and it may be useful in cytological screening of high risk patients.

Carcinoma, Squamous Cell ; diagnosis ; metabolism ; virology ; Cervical Intraepithelial Neoplasia ; diagnosis ; metabolism ; virology ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Cytodiagnosis ; DNA, Viral ; analysis ; Female ; Humans ; Immunohistochemistry ; Papillomaviridae ; genetics ; isolation & purification ; Papillomavirus Infections ; diagnosis ; metabolism ; virology ; Predictive Value of Tests ; Uterine Cervical Dysplasia ; diagnosis ; metabolism ; virology ; Uterine Cervical Neoplasms ; diagnosis ; metabolism ; virology

Methylation of p16 is an important mechanism in cervical carcinogenesis. However, the relationship between cervical squamous cell carcinoma (SCC) and Epstein-Barr virus (EBV) remains controversial. Here, we explored whether EBV infection and/or p16 gene inactivation would play any role in cervical carcinogenesis. Eighty-two specimens included 41 invasive SCCs, 30 cervical intraepithelial neoplasm (CIN; CIN 1, 11 cases, CIN II, 3 cases, CIN III 16 cases) and 11 nonneoplastic cervices. EBV was detected by polymerase chain reaction (PCR) for EBNA-1 and in situ hybridization for EBER-1. The p16 methylation-status and the expression of p16 protein were studied by methylation-specific PCR and immunohistochemistry, respectively. The materials were divided into four groups: 1) nonneoplastic cervices, 2) CIN I, 3) CIN II-III and 4) invasive SCCs. p16 methylation and p16 immunoexpressions increased in CIN and invasive SCCs than nonneoplastic tissue. p16-methylation and p16-immunoreactivities were higher in the EBV-positive group (p=0.009, p<0.001) than in the EBV-negative group. EBV was detected more frequently in CIN and SCCs than nonneoplastic cervices. In conclusion, a correlation between p16 methylation, p16 immunoreactivity and the detection of EBV strongly suggested that the cooperation of EBV and p16 gene may play a synergic effect on cell cycle deregulation.
Carcinoma, Squamous Cell/genetics/*pathology/virology ; Comparative Study ; Cyclin-Dependent Kinase Inhibitor p16/analysis/*genetics ; *DNA Methylation ; DNA, Viral/genetics/isolation & purification ; Epstein-Barr Virus Infections/genetics/*pathology/virology ; Epstein-Barr Virus Nuclear Antigens/genetics ; Female ; Herpesvirus 4, Human/genetics ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Polymerase Chain Reaction ; Precancerous Conditions/genetics/*pathology/virology ; RNA, Viral/genetics ; Research Support, Non-U.S. Gov't ; Uterine Cervical Neoplasms/genetics/*pathology/virology

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

OBJECTIVETo design DNA microarray and investigate the molecular anti-tumor mechanism of herbs of traditional Chinese medicine.

METHODcDNA microarrays consisting of 56 probes representing 24 human cell cycle genes were constructed, Four anti-hepatocarcinoma herbs including Radix Linderae, Hebra Artemisiae Annuae, Radix Amebiae, Radix Astragli, were chosen. Effects of herbs on SMMC-7721 cell cycle were observed by flow cytometry assay. Effects of herbs on cell cycle gene expression in SMMC-7721 cells were analyzed by comparing hybridization of Dig-Labeled cDNAs from herb-treated cells and cDNAs from untreated cells.

RESULTExpressions of cell cycle geneswere changed in different degrees after herbs treated. Some genes were down-regulated and some genes were up-regulated. The changes in gene expression agreed with the results of flow cytometry assay.

CONCLUSIONThe results suggest that these herbs may have effects on cell cycle and DNA damage checkpoint genes which may be the mechanism of the herbs, and DNA microarray can be used to investigate the biological function of extracts of traditional Chinese medicine.

Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Artemisia ; chemistry ; Astragalus membranaceus ; chemistry ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cyclin-Dependent Kinase 4 ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Cyclin-Dependent Kinases ; genetics ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Gene Amplification ; Gene Expression Profiling ; Genes, cdc ; drug effects ; Humans ; Lindera ; chemistry ; Lithospermum ; chemistry ; Liver Neoplasms ; metabolism ; pathology ; Oligonucleotide Array Sequence Analysis ; methods ; Plants, Medicinal ; chemistry ; Proliferating Cell Nuclear Antigen ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; cdc25 Phosphatases ; genetics ; metabolism