Pai-Mei Lin

Downregulation of circadian clock genes in chronic myeloid leukemia : Alternative methylation pattern of hPER3

Disruption of circadian rhythm is believed to play a critical role in cancer development. To gain further insights into the roles of circadian genes in chronic myeloid leukemia (CML), we analyzed peripheral blood from 53 healthy individuals and 35 CML patients for the expression of the nine circadian genes. The expression levels of hPER1, hPER2, hPER3, hCRY1, hCRY2 and hBMAL1 were significantly impaired in both chronic phase and blastic crisis of CML cases compared with those in healthy individuals (P < 0.001). Methylation studies in the promoter areas of these six genes revealed that only the CpG sites of the hPER3 gene were methylated in all of the CML patients, and the methylated CpG frequencies differed significantly in patients at blastic crisis (8.24 ± 0.73) or at chronic phase (4.48 ± 0.48). The CpG sites of the hPER2 gene were also methylated in 40% of the CML patients. No mutation was found within the coding region of hPER3 in CML cases. Our results suggest that the downregulated hPER3 expression in CML is correlated with the inactivation of hPER3 by methylation. (Cancer Sci 2006; 97: 1298–1307)

Mutation analysis of PTEN/MMAC1 in acute myeloid leukemia

Recently, a putative tumor suppressor gene, PTEN/MMAC1, has been identified at chromosome 10q23.3, which encodes a 403 amino acid dual‐specificity phosphatase containing a region of homology to tensin and auxillin. Somatic mutations of the PTEN/MMAC1 gene have been identified in a number of cancer cell lines and primary cancers. Mutations in PTEN/MMAC1 are most frequently found in advanced cancers. To evaluate the role of the PTEN/MMAC1 gene in leukemia, bone marrow and/or peripheral blood from 62 acute myeloid leukemia (AML) patients, 5 hemopoietic cell lines (HL60, U937, Raji, KG‐1, K562), and 30 normal controls were analyzed. The results showed aberrant PTEN/MMAC1 transcripts in 15 of the 62 (24%) AML patients, 4 of the 5 cell lines (80%), and 4 of the 30 (13%) normal controls. As in our previous study of TSG101, the abnormal transcripts may result from aberrant RNA splicing as evidenced by the presence of both these aberrant transcripts and normal full length transcripts in all specimens examined. Loss of heterozygosity (LOH) analysis and PCR‐SSCP of the entire coding region showed that none of the AML cases had LOH or mutation. Only one frameshift mutation at codon 130 (insertion of CCCG) with premature termination of coding sequence was observed in the U937 cell line. Our results indicate that the PTEN/MMAC1 gene may play a role in a small percentage of AML, but its significance needs to be further evaluated. Am. J. Hematol. 63:170–175, 2000.

Altered expression of SIRT gene family in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) include a group of malignant neoplasms that arise from the upper aerodigestive tract and represent the seventh most common cause of cancer-related death. The overall 5-year survival rates have not significantly improved for decades in spite of the advances in the field of oncology and surgery, encouraging further research on factors that might modify disease prognosis. The silent information regulator (SIR) genes (Sirtuins) play key roles in cellular stress and are associated with aging-related diseases including cancer. Currently, seven human sirtuin (SIRT1–7) genes have been identified, but the roles of SIRT genes in HNSCC are still uncertain. Therefore, in this study, we used real-time quantitative reverse transcription-polymerase chain reaction to investigate the expressions of the seven SIRT genes in human HNSCC tissues to assess the changes in cancerous and noncancerous parts and the correlation with different tumor behaviors. Our results demonstrated that the expression levels of SIRT1, SIRT2, SIRT3, SIRT5, SIRT6, and SIRT7 were significantly downregulated in cancerous tissues compared with noncancerous tissues (all p < 0.01). The expression levels of SIRT1, SIRT2, SIRT3, SIRT5, and SIRT7 showed downregulation in advanced stages in respect to early stages (p < 0.05). These results indicate that the downregulation of SIRT genes expression may contribute to the development of cancer and trigger the neoplastic disease to more advanced stages. Our study indicates that SIRT genes expression could help in the diagnosis and represent a prognostic biomarker in HNSCC.

Circulating miRNA is a novel marker for head and neck squamous cell carcinoma

The aim of the study is to investigate the alteration of plasma miRNA in head and neck squamous cell carcinoma (HNSCC). Altered microRNAs (miRNAs) expression has been found in many cancers, including lung cancer, breast cancer, prostate cancer, bladder cancer and colorectal cancer. Many recent studies have demonstrated that aberrant plasma miRNAs were also found in various types of cancers. However the alteration of plasma expression in HNSCC remains unclear. In this present study, the expression profiles of ten miRNAs, let-7a, miR-21, miR26b, miR-34c, miR-99a, miR-133a, miR-137, miR-184, miR-194a, and miR-375, in plasma from 50 patients and 36 healthy subjects were evaluated using real-time quantitative polymerase chain reaction (PCR). Our results demonstrated that the expression level of miR-21 was significantly up-regulated in plasma samples obtained from HNSCC patients (p < 0.01) than those from healthy subjects, which were in consistent with our finding in HNSCC tissues. A 7.7-fold increase of miR-21 in cancerous parts when compared to their non-cancerous counterparts (p < 0.0001) was observed in HNSCC tissues. In addition, the expression levels of miR-21 and miR-26b were both reduced in post-operative HNSCC patients with good prognosis. In contrast, the concentration of plasma miR-21 and miR-26b stayed high after tumor removal in the expired cases. Our study suggests that detecting circulating miR-21 and miR-26b pre- and post-operatively might provide a novel tumor marker for HNSCC.

Altered expression of circadian clock genes in human chronic myeloid leukemia.

Circadian clock genes use transcriptional-translational feedback loops to control circadian rhythms. Recent studies have demonstrated that expression of some circadian clock genes displays daily oscillation in peripheral tissues including peripheral blood and bone marrow. Circadian rhythms regulate various functions of human body, and the disruption of circadian rhythm has been associated with cancer development and tumor progression. However, the direct links between aberrant circadian clock gene expression and human disorders remain largely unknown. In this study, comparisons were made between the expression profiles of 9 circadian clock genes from peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) from 18 healthy volunteers. Peripheral blood (PB) total leukocytes from 54 healthy volunteers and 95 patients with chronic myeloid leukemia (CML) were also investigated. Similar expression profiles of all 9 circadian clock genes were observed in PBMCs and PMNs of healthy individuals. In PB total leukocytes of healthy individuals, the daily pattern of PER1, PER2, PER3, CRY1, CRY2, and CKIε expression level peaked at 0800 h, and BMAL1 peaked at 2000 h. Daily pattern expression of these 7 genes was disrupted in newly diagnosed pre—imatinib mesylate—treated and blast crisis—phase patients with CML. Partial daily pattern gene expression recoveries were observed in patients with CML with complete cytogenetic response and major molecular response. The expression of CLOCK and TIM did not show a time-dependent variation among the healthy and patients with CML. These results indicate a possible association of the disrupted daily patterns of circadian clock gene expression with the pathogenesis of CML.

Deregulated expression of circadian clock genes in gastric cancer

BackgroundGastric cancer (GC), an aggressive malignant tumor of the alimentary tract, is a leading cause of cancer-related death. Circadian rhythm exhibits a 24-hour variation in physiological processes and behavior, such as hormone levels, metabolism, gene expression, sleep and wakefulness, and appetite. Disruption of circadian rhythm has been associated with various cancers, including chronic myeloid leukemia, head and neck squamous cell carcinoma, hepatocellular carcinoma, endometrial carcinoma, and breast cancer. However, the expression of circadian clock genes in GC remains unexplored.MethodsIn this study, the expression profiles of eight circadian clock genes (PER1, PER2, PER3, CRY1, CRY2, CKIϵ, CLOCK, and BMAL1) of cancerous and noncancerous tissues from 29 GC patients were investigated using real-time quantitative reverse-transcriptase polymerase chain reaction and validated through immunohistochemical analysis.ResultsWe found that PER2 was significantly up-regulated in cancer tissues (p < 0.005). Up-regulated CRY1 expression was significantly correlated with more advanced stages (stage III and IV) (p < 0.05).ConclusionsOur results suggest deregulated expressions of circadian clock genes exist in GC and circadian rhythm disturbance may be associated with the development of GC.

Induction of Cellular Senescence by Doxorubicin Is Associated with Upregulated miR-375 and Induction of Autophagy in K562 Cells

Background Cellular senescence is a specialized form of growth arrest that is generally irreversible. Upregulated p16, p53, and p21 expression and silencing of E2F target genes have been characterized to promote the establishment of senescence. It can be further aided by the transcriptional repression of proliferation-associated genes by the action of HP1γ, HMGA, and DNMT proteins to produce a repressive chromatin environment. Therefore, senescence has been suggested to functions as a natural brake for tumor development and plays a critical role in tumor suppression and aging. Methodology/Principal Findings An in vitro senescence model has been established by using K562 cells treated with 50 nM doxorubicin (DOX). Since p53 and p16 are homozygously deleted in the K562 cells, the DOX-induced senescence in K562 cells ought to be independent of p53 and p16-pRb pathways. Indeed, no change in the expression of the typical senescence-associated premalignant cell markers in the DOX-induced senescent K562 cells was found. MicroRNA profiling revealed upregulated miR-375 in DOX-induced senescent K562 cells. Treatment with miR-375 inhibitor was able to reverse the proliferation ability suppressed by DOX (p<0.05) and overexpression of miR-375 suppressed the normal proliferation of K562 cells. Upregulated miR-375 expression was associated with downregulated expression of 14-3-3zeta and SP1 genes. Autophagy was also investigated since DOX treatment was able to induce cells entering senescence and eventually lead to cell death. Among the 24 human autophagy-related genes examined, a 12-fold increase of ATG9B at day 4 and a 20-fold increase of ATG18 at day 2 after DOX treatment were noted. Conclusions/Significance This study has demonstrated that in the absence of p53 and p16, the induction of senescence by DOX was associated with upregulation of miR-375 and autophagy initiation. The anti-proliferative function of miR-375 is possibly exerted, at least in part, by targeting 14-3-3zeta and SP1 genes.

Aberrant FHIT transcripts in acute myeloid leukaemia

Recently the FHIT gene (fragile histidine triad gene) has been identified at chromosome 3p14.2 and a high frequency of abnormalities in this gene has been demonstrated in various cancers. To determine the role of the FHIT gene in leukaemia, bone marrow or peripheral blood from 62 acute myeloid leukaemia patients and five haemopoietic cell lines (HL60, U937, Raji, KG‐1, K562) were analysed by reverse transcription of the FHIT mRNA followed by PCR amplification and sequencing of the products. To detect the deletion of the FHIT gene, 17 cases were evaluated using microsatellite polymorphism analysis. In this study, 17/62 (27%) AML patients expressed aberrant transcripts which lack two or more exons of the FHIT gene, and all the cell lines exhibited the aberrant FHIT transcripts. No cases exhibited a loss of the FHIT alleles. Our data indicated that the FHIT gene may play a role in myeloid carcinogenesis and may be indicated in the late progression of the disease.

Aberrant TSG101 transcripts in acute myeloid leukaemia

Recently, a tumour susceptibility gene, TSG101, has been identified at chromosome 11p15. A large intragenic deletion of this gene has been demonstrated in primary breast tumours. To evaluate the role of the TSG101 gene in leukaemia, bone marrow and/or peripheral blood from 68 acute myeloid leukaemia patients, five haemopoietic cell lines (HL60, U937, Raji, KG‐1, K562) and 30 normal controls were analysed by reverse transcription of the TSG101 mRNA, followed by PCR amplification and sequencing of the products. The results showed aberrant TSG101 transcripts in 24/68 (35%) acute myeloid leukaemia (AML) patients, all of the cell lines (100%) and 3/30 (10%) normal controls. Our study indicated that the abnormal transcripts may have resulted from aberrant RNA splicing as evidenced by these aberrant transcripts. Also, normal full‐length transcripts were present in all specimens examined. The aberrant transcript occurred more frequently in the AML and cell lines. However, because aberrant transcripts of TSG101 were also found in the normal controls, the role of TSG101 as a tumour suppressor gene should be evaluated carefully.

Herpes zoster is associated with an increased risk of subsequent lymphoid malignancies - A nationwide population-based matched-control study in Taiwan

BackgroundInfectious agents have been shown to contribute to the development of lymphoid malignancies. The different distribution of lymphoid malignancies in Asian and Western populations suggests possibly different etiologies in Asian populations. Herpes zoster infection, commonly seen in immunocompromised persons, has been reported to be associated with lymphoid malignancies in retrospective case–control studies from Western populations, but the results are controversial and large-scale prospective studies from Asian populations are lacking.MethodsA nationwide population-based matched-controlled prospective study on Taiwanese patients was performed using the National Health Insurance Research Database from 1996 to 2007. Herpes zoster and malignancies were defined by compatible ICD-9-CM (International Classification of Disease, 9th Revision, Clinical Modification) codes. Patients who had been diagnosed with any malignancies before herpes zoster, with known viral infections including human immunodeficiency virus, and duration from herpes zoster to diagnosis of malignancies less than 6 months were excluded.ResultsOf 42,498 patients with herpes zoster prior to the diagnosis of any malignancies, the cumulative incidence for lymphoid malignancies was 0.11% (n = 48), compared with 0.06% (n = 106) in 169,983 age- and gender-matched controls (univariate hazard ratio (HR): 1.82, 95%CI: 1.29-2.55). The most common lymphoid malignancy was non-Hodgkin’s lymphoma (60.4%, n = 29), followed by multiple myeloma (27.1%, n = 13). Risk for developing lymphoid malignancies is significantly higher in herpes zoster patients (log rank P = 0.005). After adjusting for presence of any comorbidities in Charlson comorbidity index, time-dependent covariate for herpes group, and income category using Cox proportional hazard regressions, herpes zoster patients had an increased risk of developing lymphoid malignancies (adjusted HR: 1.68, 95%CI: 1.35-2.42, P = 0.0026), but did not have an increased risk of developing non-lymphoid malignancies (adjusted HR: 1.00, 95%CI: 0.91-1.05, P = 0.872).ConclusionPreceding herpes zoster infection is an independent risk marker for subsequent lymphoid malignancies in Taiwanese subjects. Further studies are warranted for pathogenesis exploration and preventive strategies in Asian populations.