Methylation analysis of asparagine synthetase gene in acute lymphoblastic leukemia cells.
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Aspartate-Ammonia Ligase↗
Biomedical subjects
C W Miller
Publications and source records attributed to C W Miller.
DLK1: increased expression in gliomas and associated with oncogenic activities.
DLK1 (delta-like) is a transmembrane and secreted protein in the epidermal growth factor-like homeotic family. Although expressed widely during embryonic development, only a few tissues retain the expression in adults. Neuroendocrine tumors often highly express this protein; therefore, we hypothesized that brain tumors might also express it. This study found that the expression of DLK1 in gliomas was higher than that in normal brain (P < 0.05). After stable transfection of a DLK1 cDNA expression vector into GBM cell lines, their proliferation was increased. Furthermore, they lost contact inhibition, had enhanced anchorage-independent growth in soft agar, and had significantly greater capacity to migrate. Western blot studies showed that expression of cyclin D1, CDK2, and E2F4 were increased, and Rb levels were decreased in these cells. DLK1 was found on the cell surface and secreted in the medium from the transfected GBM cells. DLK1-enriched condition medium stimulated the growth of glioblastoma multiforme cell lines and explants. DLK1 antibody blocked cell growth stimulated by DLK1. In summary, these results suggest that DLK1 may play a role in the formation or progression of gliomas.
Blotting, Western↗
Dlk1 in normal and abnormal hematopoiesis.
Dlk1 (Pref-1) is a transmembrane and secreted protein, which is a member of the epidermal growth factor-like family, homologous to Notch/Delta/Serrate. We have found by real-time RT-PCR that Dlk1 mRNA levels were high in CD34(+) cells in 10 of 12 MDS samples compared with CD34(+) cells from 11 normals. Also, Dlk1 mRNA was elevated in mononuclear, low density bone marrow cells from 11/38 MDS patients, 5/11 AML M6 and 2/4 AML M7 samples. Furthermore, 5/6 erythroleukemia and 2/2 megakaryocytic leukemia cell lines highly expressed Dlk1 mRNA. Levels of Dlk1 mRNA markedly increased during megakaryocytic differentiation of both CMK megakaryoblasts as well as normal CD34(+) hematopoietic stem cells. High serum levels of Dlk1 occurred in RA (4/10) and essential thrombocythemia (2/10) patients. Functional studies showed that forced expression of Dlk1 enhanced proliferation of K562 cells growing in 1% fetal bovine serum. Analysis of hematopoiesis of Dlk1 knockout mice suggested that Dlk1 contributed to granulocyte, megakaryocyte and B-cell clonogenic growth and was needed for generation of splenic B-cells. In summary, Dlk1 is overexpressed in selected samples of MDS (especially RA and RAEB) and AML (particularly M6, M7), and it appears to be associated with normal development of megakaryocytes and B cells.
Animals↗
Triboelectric simulation of pacemaker malfunction.
We report the occurrence of a triboelectric phenomenon (static electricity) that mimicked malfunction of a contemporary pacemaker by creating an electrocardiograpic artifact virtually identical to the pacemaker stimuli. The diagnosis was established by observing a subtle overshoot of the questionable deflection that was absent from pacemaker stimuli.
Aged↗
Cyr61, a member of CCN family, is a tumor suppressor in non-small cell lung cancer.
Cysteine-rich protein 61 (Cyr61) is a member of a family of growth factor-inducible immediate-early genes. It regulates cell adhesion, migration, proliferation, and differentiation and is involved in tumor growth. In our experiments, the role of Cyr61 in non-small cell lung cancer (NSCLC) was examined. Expression of Cyr61 mRNA was decreased markedly in four of five human lung tumor samples compared with their normal matched lung samples. NSCLC cell lines NCI-H520 and H460, which have no endogenous Cyr61, formed 60-90% fewer colonies after being transfected with a Cyr61 cDNA expression vector than cells transfected with the same amount of empty vector. After stable transfection of a Cyr61 cDNA expression vector, proliferation of both H520-Cyr61 and H460-Cyr61 sublines decreased remarkably compared with the cells stably transfected with empty vector. The addition of antibody against Cyr61 partially rescued the growth suppression of both H520-Cyr61 and H460-Cyr61 cells. Cell cycle analysis revealed that both H520-Cyr61 and H460-Cyr61 cells developed G(1) arrest, prominently up-regulated expression of p53 and p21(WAF1), and had decreased activity of cyclin-dependent kinase 2. The increase of pocket protein pRB2/p130 was also detected in these cells. Notably, both of the Cyr61-stably transfected lung cancer cell lines developed smaller tumors than those formed by the wild-type cells in nude mice. Taken together, we conclude that Cyr61 may play a role as a tumor suppressor in NSCLC.
Animals↗
Mutational analysis of the peroxisome proliferator-activated receptor gamma gene in human malignancies.
Peroxisome proliferator-activated receptor gamma (PPARgamma) plays an important role in adipocyte differentiation and is expressed in many human malignancies, including those from prostate, breast, as well as colon. It regulates differentiation and/or cell growth of these cells. However, expression of this nuclear hormone receptor in other types of cancer, especially in hematological malignancies, remains to be fully elucidated. The PPARgamma gene has been mapped to chromosome band 3p25, where chromosomal abnormalities are observed in a variety of human malignancies. Furthermore, a recent study revealed that the PPARgamma gene is functionally mutated in sporadic colon cancer cells. Therefore, PPARgamma could be an important tumor suppressor gene. This prompted us to investigate the expression and mutational status of the PPARgamma gene in cancers of a variety of tissues. A total of 159 samples were interrogated for their expression of PPARgamma as measured by reverse transcription-polymerase chain reaction and/or Western blot analysis. In each of the samples, expression of PPARgamma was detectable. In addition, a total of 397 clinical samples and cell lines including colon, prostate, breast and lung cancers, and leukemias were analyzed for mutations of the PPARgamma gene by either reverse transcription-polymerase chain reaction-single-strand conformation polymorphism or polymerase chain reaction-single-strand conformation polymorphism analysis. No abnormalities were detectable in any of the human malignancies. On the other hand, shifted bands were easily detectable when using positive controls, which harbored the same sequence alterations reported previously in colon cancer cells. Taken together, PPARgamma is expressed in a variety of cancers, and mutation of the PPARgamma gene is a very rare event in human malignancies.
Blotting, Western↗
Mutations in the mitotic check point gene, MAD1L1, in human cancers.
Aneuploidy is a characteristic of the majority of human cancers, and recent studies suggest that defects of mitotic checkpoints play a role in carcinogenesis. MAD1L1 is a checkpoint gene, and its dysfunction is associated with chromosomal instability. Rare mutations of this gene have been reported in colon and lung cancers. We examined a total of 44 cell lines (hematopoietic, prostate, osteosarcoma, breast, glioblastoma and lung) and 133 fresh cancer cells (hematopoietic, prostate, breast and glioblastoma) for alterations of MAD1L1 by RT-PCR-SSCP and nucleotide sequencing. Eight mutations consisting of missense, nonsense and frameshift mutations were found, together with a number of nucleotide polymorphisms. All the alterations in cell lines were heterozygous. Frequency of mutations was relatively high in prostate cancer (2/7 cell lines and 2/33 tumor specimens). We placed a mutant truncated MAD1L1, found in a lymphoma sample, into HOS, Ht161 and SJSA cell lines and found that it was less inhibitory than wild type MAD1L1 at decreasing cell proliferation. Co-expression experiments showed that the mutant form had a dominant-negative effect. Furthermore, this mutant impaired the mitotic checkpoint as shown by decreased mitotic indices in HOS cells expressing mutant MAD1L1 after culture with the microtubule-disrupting agent, nocodazole. Our results suggest a pathogenic role of MAD1L1 mutations in various types of human cancer.
Breast Neoplasms↗
Tumor necrosis factor alpha polymorphism associated with increased susceptibility to development of adult T-cell leukemia/lymphoma in human T-lymphotropic virus type 1 carriers.
Human T-lymphotropic virus type 1 (HTLV-1) is etiologically associated with adult T-cell leukemia/lymphoma (ATL). Nevertheless, most individuals infected with HTLV-1 do not develop ATL. To attempt to identify genetic factors promoting the progression to ATL, we investigated in HTLV-1 carriers the relationship between susceptibility to ATL and several polymorphisms: the three "decreased-detoxifying" polymorphisms in GSTM1, GSTT1, and CYP1A1, the "proapoptotic" polymorphism in BCL2, and the five "high-production" polymorphisms in tumor necrosis factor alpha (TNF-alpha) using PCR-based genotyping assays. ATL patients (n = 71) were younger than HTLV-1 carriers (n = 80; 57 +/- 12 versus 63 +/- 10 years; P = 0.0017). MALE:female ratio in ATL patients was higher than in carriers (52:19 versus 19:61, respectively; P < 0.0001), probably reflecting a higher incidence of HTLV-1 infection in females and a higher incidence of development of ATL in males. We found that the frequency of the TNF-alpha-857T allele, reported to be associated with high transcriptional activity of the promoter/enhancer region of the TNF-alpha gene, was enriched in individuals with ATL compared with healthy carriers (18.3% versus 8.8%, respectively; odds ratio, 2.34; 95% confidence interval, 1.2-4.7). None of the other four TNF-alpha polymorphisms was a significant indicator of risk of development of ATL, although odds ratios (ATL versus carrier) of all of the TNF-alpha polymorphisms were higher than 1.0. Furthermore, analysis of polymorphisms for GSTM1, GSTT1, CYP1A1, and BCL2 showed no significant difference between ATL patients and healthy carriers. Genetic polymorphism leading to increased TNF-alpha production may enhance susceptibility to ATL among HTLV-1 carriers. Alternatively, but less likely, the HLA loci might be an important factor because the TNF-alpha gene lies within the class III region of the MHC; however, the 857T allele is not in linkage disequilibrium with HLA alleles associated with ATL development.
Carrier State↗
Breast cancer. Cyr61 is overexpressed, estrogen-inducible, and associated with more advanced disease.
To identify genes involved in breast cancer, polymerase chain reaction-selected cDNA subtraction was utilized to construct a breast cancer-subtracted library. Differential screening of the library isolated the growth factor-inducible immediate-early gene Cyr61, a secreted, cysteine-rich, heparin binding protein that promotes endothelial cell adhesion, migration, and neovascularization. Northern analysis revealed that Cyr61 was expressed highly in the invasive breast cancer cell lines MDA-MB-231, T47D, and MDA-MB-157; very low levels were found in the less tumorigenic MCF-7 and BT-20 breast cancer cells and barely detectable amounts were expressed in the normal breast cells, MCF-12A. Univariate analysis showed a significant or borderline significant association between Cyr61 expression and stage, tumor size, lymph node positivity, age, and estrogen receptor levels. Interestingly, expression of Cyr61 mRNA increased 8- to 12-fold in MCF-12A and 3- to 5-fold in MCF-7 cells after 24- and 48-h exposure to estrogen, respectively. Induction of Cyr61 mRNA was blocked by tamoxifen and ICI182,780, inhibitors of the estrogen receptor. Stable expression of Cyr61 cDNA under the regulation of a constitutive promoter in MCF-7 cells enhanced anchorage-independent cell growth in soft agar and significantly increased tumorigenicity and vascularization of these tumors in nude mice. Moreover, overexpression of Cyr61 in MCF-12A normal breast cells induced their tumor formation and vascularization in nude mice. In summary, these results suggest that Cyr61 may play a role in the progression of breast cancer and may be involved in estrogen-mediated tumor development.
Adult↗
Simian virus 40 is present in human lymphomas and normal blood.
Many independent studies have demonstrated Simian virus 40 (SV40) in normal and neoplastic human tissues. Clonal integration of virus in the DNA of several thyroid and bone tumors suggests a direct role for SV40 in some cancers. However, in most cases the role of SV40 remains unclear. This study determined the presence of SV40, by amplification followed by hybridization, in 266 normal and neoplastic blood and lymphoid samples. Amplification detected SV40 in 14% of non-autoimmune deficiency syndrome (AIDS) lymphomas, 28% of AIDS related lymphoma and 16% of peripheral blood lymphocytes from non-cancerous patients. No SV40 was detected in leukemia samples. Direct Southern blotting of SV40+ samples detected no virus, consistent with less than one viral genome in ten cells. Sequence analysis of SV40 in blood and lymphoid samples found sequences distinct from laboratory strains of SV40. The presence of limited quantities of SV40 in a small proportion of both normal and neoplastic tissues is suggestive of an adventitious presence with no apparent direct role in blood and lymphoid cancers.
Antigens, Polyomavirus Transforming↗
Dietary chloride as a possible determinant of the severity of exercise-induced asthma.
Dietary sodium chloride (NaCl) has been shown to alter the severity of exercise-induced asthma, but it is not known if the sodium and chloride ions have independent effects in this regard. The hypothesis tested in the present study was that both a low sodium, low chloride diet and a high sodium, low chloride diet would improve post-exercise pulmonary function in subjects with exercise-induced asthma (EIA) compared to a normal NaCl diet (NSD); but that neither of these diets would have an effect on post-exercise pulmonary function in control (non-EIA) subjects. Eight subjects who suffered from EIA and eight subjects who did not (control) took part in a double-blind crossover study. Pre- and post-exercise pulmonary function was assessed after 2 weeks on a NSD, a low NaCl diet (LSD, low sodium, low chloride) or a sodium bicarbonate diet (NaHCO3 diet, high sodium, low chloride). A 1 week washout period occurred between diets. Altering dietary sodium or chloride had no effect on pre-exercise (baseline) pulmonary function in either group or on post-exercise pulmonary function in control subjects. However, both the LSD and the NaHCO3 diet lessened the deterioration in post-exercise pulmonary function in EIA subjects. Comparing results from pre- to post-exercise, forced expiratory volume in 1 s (FEV1) at 15 min post-exercise differed significantly (P < 0.05) between diets [mean (SEM) 7 (4)% on the LSD, 14 (4)% on the NaHCO3 diet, and 19 (2)% on the NSD]. Similar patterns were observed for forced vital capacity (FVC), forced expiratory flow rate at 25%-75% FVC and peak expiratory flow rate. The NaHCO3 diet lessened the deterioration of post-exercise pulmonary function, but not to the extent of LSD. These data suggest that both sodium and chloride contribute to the worsening of EIA symptoms seen after consuming a normal or high NaCl diet.
Acidosis↗
Mutation analysis of the DNA-damage checkpoint gene CHK2 in myelodysplastic syndromes and acute myeloid leukemias.
Checkpoint genes code for a family of proteins which sense DNA damage in eukaryotic cells. They play an important role in the control of the cell cycle. The human CHK2 is a homolog of the yeast G(2) checkpoint kinases known as CDS1 and RAD53. The CHK2 may be a tumor suppressor gene because it was found to be mutated in some individuals with the Li-Fraumeni syndrome. These cases had a normal, non-mutated p53 gene. We performed a mutational analysis of the CHK2 gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) in 41 bone marrow samples from individuals with myelodysplastic syndrome (MDS) and 41 samples of acute myeloid leukemias (AML). We found a novel G to C transversion resulting in a change from Ala to Gly at codon 507 of CHK2 in one MDS sample, but normal cells from this individual did not have the abnormality. In addition, we demonstrated a previously described polymorphism at codon 84 (A to G at nucleotide 252) of exon 1 of CHK2 in three of 41 MDS and three of 41 AML patients. The presence of a CHK2 mutation in MDS highlights the importance of alterations of cell cycle checkpoint genes in this disease.
Acute Disease↗
Frequent loss of heterozygosity in the region of D1S450 at 1p36.2 in myelodysplastic syndromes.
To understand the underlying mechanisms in myelodysplastic syndromes (MDS) by identifying target tumor suppressor genes, we performed a detailed deletional mapping of the short arm of chromosome 1 in 38 paired samples of bone marrow and peripheral blood obtained from individuals with MDS by PCR amplification of a total of 23 highly informative microsatellite sequences. We identified the commonly deleted region between D1S508 and D1S244. LOH of this region was found in five patients (13%). In addition, LOH at 1p was associated with a poor clinical outcome, suggesting that the deletion of a gene in this region may be involved in the course of this disease. By analyzing the chromosomal map of this region, we found TNFRSF12 as a candidate tumor suppressor gene. However, our search for mutations in this gene did not identify somatic mutations in MDS. Our findings are consistent with the possible existence of an as-yet unknown tumor suppressor gene in this region that is altered in MDS.
Adult↗
Long-term clinical and radiographic results of a pegged tibial baseplate in primary total knee arthroplasty.
Tibial baseplates of total knee arthroplasty components use stems, fins, and pegs to augment fixation. A pegged design without a stem offers flexibility in tray orientation and ease of revision. These advantages would not be important if stemless designs resulted in a higher rate of aseptic loosening. This article presents an average 10.5-year follow-up of a system offering a pegged tibial baseplate. Good or excellent results in 91% of patients are cited with a revision rate for aseptic loosening of 1%. A pegged design may offer advantages without a higher rate of complications when compared with stemmed implants.
Arthroplasty, Replacement, Knee↗
Closed reduction of a dislocation of a constrained acetabular component.
A constrained acetabular liner occasionally is used in management of chronic instability after total hip arthroplasty. If dislocation occurs out of a constrained liner, open reduction is indicated. A case is presented of closed reduction of a femoral component into a constrained liner.
Aged↗
Evaluation of accuracy and reliability of indirect calorimetry for the measurement of resting energy expenditure in healthy dogs.
OBJECTIVE: To assess accuracy and reliability of open-flow indirect calorimetry in dogs. ANIMALS: 13 clinically normal dogs. PROCEDURE: In phase 1, oxygen consumption per kilogram of body weight (VO2/kg) was determined in 6 anesthetized dogs by use of open-flow indirect calorimetry before and after determination of VO2/kg by use of closed-circuit spirometry. In phase 2, four serial measurements of VO2 and carbon dioxide production (VCO2) were obtained in 7 awake dogs by use of indirect calorimetry on 2 consecutive days. Resting energy expenditure (REE) was calculated. RESULTS: Level of clinical agreement was acceptable between results of indirect calorimetry and spirometry. Mean VO2/kg determined by use of calorimetry before spirometry was significantly greater than that obtained after spirometry. In phase 2, intraclass correlation coefficients (ICC) for REE and VO2 were 0.779 and 0.786, respectively, when data from all 4 series were combined. When the first series was discounted, ICC increased to 0.904 and 0.894 for REE and VO2, respectively. The most reliable and least variable measures of REE and VO2 were obtained when the first 2 series were discounted. CONCLUSIONS AND CLINICAL RELEVANCE: Open-flow indirect calorimetry may be used clinically to obtain a measure of VO2 and an estimate of REE in dogs. Serial measurements of REE and VO2 in clinically normal dogs are reliable, but a 10-minute adaption period should be allowed, the first series of observations should be discounted, multiple serial measurements should be obtained, and REE.
Animals↗
Detection of free radicals in ischemic and reperfused canine gracilis muscle flaps by use of spin-trapping electron paramagnetic resonance spectroscopy.
OBJECTIVE: To determine whether free radicals are produced in ischemic and reperfused canine skeletal muscle, whether free radicals can be detected from effluent blood by use of spin-trapping electron paramagnetic resonance (EPR) spectroscopy, and whether free radical-induced skeletal muscle damage is detectable by use of light microscopy. ANIMALS: 6 healthy mixed-breed dogs. PROCEDURES: Dogs were anesthetized and both gracilis muscles were isolated, leaving only the major vascular pedicle intact. Ischemia was induced in 1 flap for 4 hours; the contralateral flap served as the control. Ischemic flaps were then reperfused for 15 minutes. alpha-Phenyl-N-tert-butylnitrone, a spin-trapping agent, was administered intravenously to each dog 1 hour prior to reperfusion. Following reperfusion, effluent blood samples from muscle flaps were obtained and processed for EPR spectroscopy. Muscle biopsy specimens were obtained for histologic evaluation, and dogs were euthanatized. RESULTS: Spin adducts were not detected in blood from control flaps. However, spin adducts were detected in all ischemic-reperfused muscle flaps. Principal signals identified were characteristic of oxygen- and carbon-centered radicals. Significantly more muscle damage was detected in ischemic-reperfused flaps, compared with control flaps. CONCLUSIONS AND CLINICAL RELEVANCE: Free radicals may be an important component of injury induced by ischemia and reperfusion of canine skeletal muscle. Spin-trap adducts of free radicals can be detected in effluent blood of canine muscle flaps by use of spin-trapping EPR spectroscopy. Spin-trapping EPR spectroscopy may be useful for the study of antioxidants and free radical scavengers in attenuating ischemia and reperfusion-mediated skeletal muscle damage.
Animals↗
Analysis of the CHK2 gene in lymphoid malignancies.
The CHK2 gene encodes a protein kinase that is important for the regulation of cell cycle arrest after DNA damage. CHK2 acts downstream of ataxia teleangiecstasia mutated (ATM), modulates the function of p53 and may help mediate cell cycle arrest at G2/M by phosphorylation of Cdc25C. Recently, the human homolog of the checkpoint kinase Cds1 (CHK2) has been suggested to be a tumor suppressor gene. Heterozygous germline mutations have been reported in Li-Fraumeni syndrome (LFS), a highly penetrant familial cancer phenotype, and in sporadic colon cancer. LFS is associated with the development of lymphoid malignancies, especially childhood ALL. Therefore, we analyzed the DNA from 143 lymphoid malignancies to determine whether they had mutations of the CHK2 gene. The 14 exons of CHK2 were studied by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and sequencing of aberrantly migrating bands. One missense mutation changing serine to phenylalanine (codon 428) in an evolutionarily highly conserved domain was found in a non-Hodgkin's aggressive lymphoma. Another point mutation in the non-coding region was identified in one of adult T-cell leukemias (ATL) samples. This result suggests that mutation of the CHK2 gene may rarely be involved in the development of selected lymphomas.
Adult↗