Hui-Hua Hsiao

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)

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.

Dihydropyrimidine dehydrogenase pharmacogenetics in the Taiwanese population.

Background/purpose5-Fluorouracil (5-FU) remains the most frequently used chemotherapy agent in various human cancers. Over 80% of the 5-FU administered is metabolized by dihydropyrimidine dehydrogenase (DPD) in the liver. However, mutations in the DPD gene have been found to be associated with low DPD activity causing severe complications. The aim of this study was to determine the frequency of 11 known mutations in Taiwanese subjects and the relationship between mutation and DPD level.MethodsSamples from a total of 300 subjects were investigated in this study. The PCR-RFLP method was used to identify 11 mutations of the DPYD gene, including 62G>A, 74A>G, 85T>C (DPYD*9A), 812delT, 1003G>T, 1156G>T, 1627A>G (DPYD*5), 1714C>G, 1897delC (DPYD*3), 2194G>A (DPYD*6), and IVS14+1G>A (DPYD*2A). DPD protein levels were determined using a DPD ELISA kit.ResultsFour mutations, including 74A>G, 85T>C (DPYD*9A), 1627A>G (DPYD*5), and 2194G>A (DPYD*6), were found in our 300 samples. The following mutations were not detected: 62G>A, 812delT, 1003G>T, 1156G>T, 1714C>G, 1897delC (DPYD*3), and IVS14+1G>A (DPYD*2A). The phenotype analysis by DPD protein level indicated that the 1627A>G (DPYD*5) mutation was not associated with the DPD protein level and might be a polymorphism in the DPD gene. The DPD level was also not correlated with gender.ConclusionNo significant correlations between these 11 mutations and DPD protein level were found indicating that examination of these mutations is insufficient to provide a high-value prediction of the 5-FU pharmacogenetic syndrome in Taiwanese. Genotype and phenotype analysis indicated the 1627A>G (DPYD*5) mutation to be a polymorphism.

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.

Urinary neutrophil gelatinase-associated lipocalin levels predict cisplatin-induced acute kidney injury better than albuminuria or urinary cystatin C levels

Cisplatin‐induced acute kidney injury (AKI) is a major concern among clinicians in prescribing cisplatin‐based chemotherapy. This study evaluated and compared the ability of urinary biomarkers, including urinary neutrophil gelatinase‐associated lipocalin (NGAL), cystatin C, and the urinary albumin to creatinine ratio (ACR) to predict cisplatin‐induced AKI. Thirty‐three cancer patients receiving cisplatin‐based chemotherapy were prospectively studied, including 10 (30%) who developed AKI (the study group). Changes of urinary biomarkers were compared at 4 hours, 8 hours, and 12 hours, and 1 day, 2 days, 3 days, and 4 days after cisplatin intravenous infusions (75 mg/m2) versus the baseline. There was a significant increase in urinary NGAL levels from 12 hours to 4 days (p < 0.05) compared to baseline after cisplatin infusion in the AKI group. The magnitude of these changes over time differed significantly by group (p < 0.001). The area under the receiver operating curve describing the relationship between urinary NGAL levels and AKI within 12 hours was 0.865 (95% confidence interval = 0.691–1.000). Urinary NGAL levels independently predicted AKI 12 hours after cisplatin (p = 0.045) after adjustments for age, gender, body mass index, baseline serum creatinine, and urinary total protein. Urinary NGAL levels may be an early biomarker of AKI in patients receiving cisplatin‐based treatment.

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.

The AGG interruption pattern within the CGG repeat of the FMR1 gene among Taiwanese population

The fragile X syndrome is the most common form of inherited mental retardation and is mainly due to an expansion of a (CGG)n trinucleotide repeat located in the 5′UTR of the fragile X mental retardation 1 (FMR1) gene at Xq27.3 (Kremer et al. 1991; Yu et al. 1991; Fu et al. 1991). In healthy individuals, the (CGG)n repeat in FMR1 is highly polymorphic, with allele sizes ranging from 5 to 50 repeats (Fu et al. 1991; Mandel and Biancalana 2004). However, two other stages of fragile X syndrome exist: the premutation stage of 50 to 200 repeats; and a full mutation, which is a massive expansion beyond 200 repeats, that causes mental retardation in males and in about 50% of female carriers. Apart from the length of the repeat, other factors, including AGG interruption pattern, length of uninterrupted CGG, and DXS548-FRAXAC1 markers, also have impact on the stability of the FMR1 gene and exert influence on the syndrome (Mandel and Biancalana 2004). The AGG interruptions in CGG triple repeat occur once in every nine or ten CGG repeats. Most normal alleles have two or more AGG interruptions in five to 50 CGG repeats, while most permutation alleles (50 to 200 repeats) have only a single or no interruption (Eichler et al. 1995; Zhong et al. 1995). The interruption is associated with certain haplotypes and is related to instability, which can lead to an expansion and eventually, the fragile X syndrome (Eichler et al. 1995; Zhong et al. 1995; Larsen et al. 2000). Because the total length of the repeat is not a clear predictor of expansion, it is important to determine both the length and the interruption pattern of the repeats to evaluate the tendency of instability. Therefore, this study was conducted to evaluate the pattern of CGG repeats in Taiwan.

Cytomegalovirus infection and disease after allogeneic hematopoietic stem cell transplantation: experience in a center with a high seroprevalence of both CMV and hepatitis B virus

Cytomegalovirus (CMV) infection and disease are important concerns after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The similarity of hepatitis B virus (HBV) and CMV with regards to their chronic viral persistence and potential reactivation at the time of impaired cellular immunity has raised clinicians’ interest in the occurrence and association between them among patients receiving allo-HSCT; however, only limited data have been obtained from a high seroprevalence region of both CMV and HBV. We monitored 117 adult allo-HSCT patients with both CMV polymerase chain reaction and pp65 antigenemia assay weekly until day 100. In 91.8% of our cases, donors and recipients were both CMV seropositive, and 13.7% of the patients were positive for HBV surface antigen. The incidences of CMV infection and disease were 45.3% and 6.8%, respectively. Grade II–IV acute graft-versus-host disease and anti-thymocyte globulin-containing conditioning regimen were associated with an increased risk of CMV infection in a multivariate analysis (hazard ratio 3.02, 95% CI 1.68–5.42, p < 0.001 and hazard ratio 5.29, 95% CI 2.57–10.8, p < 0.001). No survival disadvantage was found in patients who developed CMV infection (p = 0.699) and CMV disease (p = 0.093). No clinically significant HBV reactivation was found, and the underlying HBV infection in donors or recipients before allo-HSCT did not increase the risk of CMV infection and CMV disease and did not influence survival after allo-HSCT.

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.

Additional chromosome abnormalities in chronic myeloid leukemia

The Philadelphia (Ph) chromosome and/or Breakpoint cluster region‐Abelson leukemia virus oncogene transcript are unique markers for chronic myeloid leukemia (CML). However, CML demonstrates heterogeneous presentations and outcomes. We analyzed the cytogenetic and molecular results of CML patients to evaluate their correlation with clinical presentations and outcome. A total of 84 newly diagnosed CML patients were enrolled in the study. Patients were treated according to disease status. Bone marrow samples were obtained to perform cytogenetic and molecular studies. Clinical presentations, treatment courses, and survival were reviewed retrospectively. Among 84 patients, 72 had chronic phase and 12 had accelerated phase CML. Cytogenetic study showed 69 (82.1%) with the classic Ph chromosome, 6 (7.2%) with a variant Ph chromosome, and 9 (10.7%) with additional chromosome abnormalities. Fifty‐four (64.3%) cases harbored b3a2 transcripts, 29 (34.5%) had b2a2 transcript, and 1 had e19a2 transcript. There was no difference in clinical presentations between different cytogenetic and molecular groups; however, additional chromosome abnormalities were significantly associated with the accelerated phase. Imatinib therapy was an effective treatment, as measured by cytogenetic response, when administered as first‐ and second‐line therapy in chronic phase patients. Survival analysis showed that old age, additional chromosome abnormalities, high Sokal score, and no cytogenetic response in second‐line therapy had a significant poor impact (p < 0.05). In conclusion, we presented the cytogenetic and molecular pattern of CML patients and demonstrated that the additional chromosome abnormality was associated with poor outcome.