Leukemic White Blood Cells Research Articles

Gene expression analysis of noncoding PCA3 gene in patients with chronic myeloid leukemia

Nowadays, noncoding RNAs are of special significance to scientists. Among RNAs, long noncoding RNAs (lncRNAs) have an important role in gene expression regulation. Recent studies show aberrant lncRNAs expression in different types of cancer including blood malignancies. As such, lncRNAs could be a possible way for diagnosis and treatment of certain cancers. In the current study, the level of PCA3 gene expression in patients with chronic myeloid leukemia (CML) was compared with normal individuals to find out whether the level of PCA3 in CML patients is increased according to our hypothesis. The buffy coat was collected from peripheral blood of thirty untreated CML patients (BCR-ABL positive) and twenty normal volunteers. RNA was extracted from white blood cells and cDNA was synthesized. The level of PCA3 gene expression was measured using quantitative reverse transcription-polymerase chain reaction. The results showed that PCA3 has expression in both normal and leukemic white blood cells. The data also revealed higher expression of PCA3 in leukemic patients, compared to the normal counterpart (P < 0.05). The unusual increase in PCA3 gene expression in CML patients suggests the need for more research on mechanisms of molecular pathways related to PCA3 which could help achieve better treatment.

A Study Of Myc and Mcl-1 Transcript Levels Before and During Tyrosine Kinase Inhibitor Therapy In Chronic Myeloid Leukemia Patients

BackgroundTyrosine kinase inhibitors (TKIs) are the current treatment of choice for chronic myeloid leukemia (CML). However, approximately 15% of patients fail first and second line therapy, creating the need for the development of additional pharmacological targets in CML. Kinases acting downstream of BCR-ABL as well as anti-apoptotic proteins such as BCL-2 are being in vitro investigated as therapeutic targets. Previous research has yielded contradictory results on the role of c-myc in CML. In one study, conditional overexpression of myc was shown to confer resistance to imatinib-induced apoptosis via downregulation of the cell cycle regulator p27Kip1. In another study, myc induced p53-dependent apoptosis of leukemia-initiating cells (LICs) in CML. However, there has been so far no attempt to correlate myc transcript and protein levels with those of the bcr-abl transcript. Mcl-1, a bcl-2 family member, has been shown to promote survival of Ph+ lymphoblasts. Mcl-1 mRNA and protein levels are downregulated by imatinib in Ph + cell lines, but its expression in CML patient samples has not been investigated. AimsWe aimed to test whether expression of myc and mcl-1 is influenced by the levels of the BCR-ABL transcript and therefore compared transcript levels of these two individual genes before and during treatment with TKIs in CML patients. MethodsWe used RNA isolated from whole blood samples of thirty-six (36) patients with CML, obtained at diagnosis and during treatment with a TKI, at least three months after initiation of treatment. All samples collected during treatment had a bcr-abl/abl ratio less than 2%, while samples collected at diagnosis had bcr-abl/abl ratios ranging from 33 to 130 %. RNA samples were subjected to reverse transcription and cDNA was used in quantitative Real-Time PCR reactions employing Taqman assays for myc, mcl-1 and hprt1 (housekeeping gene). Fold induction or repression of genes after treatment was calculated via the ΔCt method, after calculation of PCR efficiencies. Statistical analysis was performed with the use of the Wilcoxon rank-sum test. ResultsMedian mRNA levels of myc were shown to be increased 2.2 times and median mRNA levels of mcl-1 were shown to be increased 2.6 times after treatment with imatinib, at a statistically significant level (p value <0.005 in both cases). There was therefore an inverse correlation of transcription of both genes with the levels of the bcr-abl transcript. ConclusionsNew treatment targets, either BCR-ABL independent or targets lying downstream of BCR-ABL, are needed in the field of CML therapy in order to circumvent the problem of TKI resistance. In our study, transcription of the genes myc and mcl-1 was shown to be moderately induced by imatinib treatment, which means that non-leukemic Ph-negative white blood cells have a higher amount of myc and mcl-1 transcripts compared to Ph+ leukemic white blood cells. Whole blood RNA was used in the study, as the BCR-ABL kinase has been shown to be expressed in the mature progeny of all haemopoietic lineages except some T cell clones. A study of the protein levels of the two genes would also be warranted in order to elucidate their potential pathogenetic or protective role in CML. In vitro functional studies with specific pharmacological inhibitors would subsequently show if the MYC and MCL-1 proteins could become targets to activate or inhibit in CML. Disclosures:No relevant conflicts of interest to declare.

How do normal and leukemic white blood cells egress from the bone marrow?

Under normal circumstances only mature granulocytes and monocytes cross the bone marrow sinus wall, a trilaminar structure consisting of endothelial cells, a discontinuous basal membrane and an adventitial cell layer in order to get access to the blood circulation. In leukemia, however, immature white blood cells are able to traverse the barrier and to appear in the blood stream. Very little is known about the regulatory processes which govern the egress of white blood cells in healthy individuals and their malignant counterparts in patients with leukemia. The results of the few studies performed to address this question in animal and human leukemias all agree that the extent to which adventitial cells (fibroblasts) cover the endothelium in bone marrow is drastically reduced. This implies altered interactions between the leukemic and adventitial cells and their extracellular matrix. We propose here a model to explain the egress of normal cells and their leukemic counterparts. It is based upon our own experimental data and the general at present limited knowledge of the subject. It is hoped that this model will stimulate further research into this important aspect of leukemogenesis.

Regulation of phosphoribosylpyrophosphate synthetase by endogenous purine and pyrimidine compounds and synthetic analogs in normal and leukemic white blood cells

Phosphoribosylpyrophosphate (PRPP) is essential for the formation of both purine and pyrimidine nucleotides as well as for the active nucleotide form of some chemotherapeutic agents. The formation of PRPP is catalyzed by the enzyme PRPP synthetase, and many different compounds are known to affect the activity of this enzyme. This report examines the effects of endogenous purine and pyrimidine nucleotides, nucleosides, and several analogs of these compounds on the activity of PRPP synthetase from different types of normal and leukemic white blood cells (i.e. normal lymphocytes, normal granulocytes, phytohemagglutinin-stimulated lymphocytes, and acute and chronic leukemic cells). Our results show that the effect varied with each individual compound, and the magnitude of the effect was dependent on the source of the enzyme. Since it appears possible to differentially affect PRPP synthetase activity from the different types of leukemic cells, this enzyme may be a potential target site in the chemotherapy of leukemia.

Multiple forms of prpp synthetase in human leukemic white blood cells seen with isoelectric focusing

1. 1. This paper presents the first description of the isoelectric points (pI) of phosphoribosyl pyrophosphate synthetase activity from normal human lymphocytes and granulocytes, PHA-stimulated normal lymphocytes and leukemic white blood cells. 2. 2. The pI values of the enzyme activities from each type of stimulated or leukemic cell were different from the pI values of the normal controls. 3. 3. Different pI values, and therefore different physical characteristics, of these active enzyme forms indicate that it might be possible to differentially inhibit the variant enzyme forms and possibly to decrease the synthesis of ribonucleotides in leukemic vs normal cells.

Comparison of the properties of phosphoribosylpyrophosphate synthetase in normal and leukemic human white blood cells

Phosphoribosylpyrophosphate synthetase (PRPP synthetase) from normal human lymphocytes and granulocytes was compared with phytohemagglutinin (PHA)-stimulated lymphocytes and leukemic white blood cells of several types with respect to enzyme activity and kinetic constants. PRPP synthetase activity was determined by measuring the production of 14CO 2 in a coupled reaction with ¦ 14C¦orotic acid in the presence of orotidylate pyrophosphorylase and orotidylate decarboxylase. Enzyme activity was expressed both as nmoles/mg of protein/30 min and as nmoles/10 3 cells/30 min. In more than 50 per cent of the leukemic patients, activity was above normal values when compared on a per mg protein basis. However, when activity was compared on a per cell basis, only the acute myelocytic leukemic patients showed a change from the normal value, and in this case activity was inversely related to the percentage of blast cells in the peripheral circulation. Michaelis constants for ATP ( K m ATP) and ribose-5-phosphate ( K m R5P) were found to be 17.6 ± 1.8 and 51.3 ± 2.4 μM, respectively, in normal lymphocytes, and 55.9 ± 8.0 and 82.5 ± 1.1 μM in normal granulocytes. The K m ATP was found to decrease in all leukemic cell types, while the K m R5P showed deviation from normal values depending on the type of leukemia. The inhibition constants ( K i ATP and K i R5P) were also compared in all leukemic cell types and showed deviations from normal which were cell type dependent. These results suggest that sufficient alteration of the properties of PRPP synthetase from leukemic cells exists to warrant further investigation into the therapeutic implications of alterations of the properties of this enzyme.

Cytoskeleton regulates β-adrenergic hormonal stimulation in normal and leukemic white blood cells

Cytoskeleton regulates β-adrenergic hormonal stimulation in normal and leukemic white blood cells

Isolation and characterization of a ribonuclease from human leukemic blood cells specific for ribonucleic acid of ribonucleic acid-deoxyribonucleic acid hybrid molecules

A ribonuclease that specifically hydrolyzes RNA in RNA. DNA hybrids has been purified more than 100-fold from human acute leukemic white blood cells. The molecular weight of this enzyme has been estimated as 80,000 by glycerol gradient centrifugation. It requires Mg-2plus for activity and is inhibited by N-ethylmaleimide. The optimum activity is observed at pH 8 (37 DEGREES). It is a heat-labile protein, t 1/2 at 50 degrees being 2 min. Among the substrates examined, (A)n X (dT)m, (I)n X (DC)m, and PHIX-174 DNA X RNA were hydrolyzed efficiently. (U)n X (dA)m showed a slight substrate activity, while (c) n X (dG) m and (G)n X (dC)m were not significantly hydrolyzed. The enzyme is an endonuclease and does not require RNA ends in the substrate molecule. It is capable of converting more than 95% of the RNA portions in hybrid substrates into acid-soluble products which are mono- and oligonucleotides terminated in 3'-OH and 5'-phosphate.

Systematics of RNA tumor viruses and virus-like particles of human origin.

[(3)H]DNA copies of avian, feline, murine, and primate RNA tumor virus genomes were synthesized in vitro by an RNA-dependent DNA polymerase reaction. These DNAs were hybridized to 60-70S RNA that had been purified from the viruses. The amount of the [(3)H]DNA hybridized yielded a measure of the genetic relatedness among the DNA preparations synthesized by the viruses. When many combinations of DNA and RNA were analyzed, the pattern of hybridization showed in some cases that the DNA copies of the viral RNA were related to each other in the same way that the natural hosts of the viruses are phylogenetically related. This pattern was observed only among the RNA leukemia viruses. The sarcoma component in sarcoma-leukemia viruses from rats and primates appeared to be unusually closely related. The mouse mammary carcinoma virus and two unclassified viruses (MPMV and Visna) appeared to be genetically distinct.A similar analysis of DNA synthesized by an RNA-dependent DNA polymerase associated with a viral-like particle obtained from the cytoplasm of human leukemic white blood cells demonstrated that this DNA occupied a space in the affinity pattern of leukemia viruses which is expected of a nucleic acid from a primate-type-C RNA tumor virus. This observation strengthens earlier evidence that components of RNA tumor viruses are associated with human leukemia.

Sequences present in both human leukemic cell nuclear DNA and Rauscher leukemia virus.

DNA synthesized by particulate fractions from human leukemic white blood cells has been subfractionated by hybridization to Rauscher leukemia virus 70S RNA followed by hydroxylapatite chromatography. The Rauscher-leukemia-virus-specific DNA fraction is shown to be complementary only to sequences present in the nuclear DNA of Balb/c mouse spleens infected by this virus and the nuclear DNA from human leukemic white blood cells, and not to sequences present in the nuclear DNA of normal Balb/c mouse spleens or the nuclear DNA of normal human white blood cells.

43. An animal model for the study of frozen human normal and leukemic white blood cells: Albert Roy, Isaac Djerassi,* Sidney Farber,* Raymond Vaillancourt,* and John Cavins (Children's Cancer Research Foundation and The Department of Pathology, Harvard Medical School, at The Children's Hospital, Boston, Massachusetts)

43. An animal model for the study of frozen human normal and leukemic white blood cells: Albert Roy, Isaac Djerassi,* Sidney Farber,* Raymond Vaillancourt,* and John Cavins (Children's Cancer Research Foundation and The Department of Pathology, Harvard Medical School, at The Children's Hospital, Boston, Massachusetts)

Methionine-activating enzyme and catechol-O-methyl transferase activity in normal and leukemic white blood cells.

IT has been found recently in these laboratories that the leucocytes of patients with chronic myelocytic leukaemia have significant elevations of the important methyl donor, S-adenosylmethionine (SAMe) (ref. 1).

ADENOSYLMETHIONINE ELEVATION IN LEUKEMIC WHITE BLOOD CELLS.

White blood cell preparations from patients with chronic myelocytic leukemia have more S-adenosylmethionine, an important metabolic intermediate, than normal peripheral white cells or thoracic-duct lymphocytes. The previously reported elevation of cyanocobalamine (vitamin B(l2)) in the serum of patients with this disease is corroborated. The possible usefulness of methylation antimetabolites in cancer chemotherapy is suggested.

Leukocyte Changes During Acute Cross-Circulation Experiments Between Leukemic and Normal or Irradiated Rats

Abstract 1. Normnal and irradiated rats underwent periods of cross-circulation with Shay granulocytic chloroleukemic rats. 2. The leukemic white blood cells disappeared rapidly from the arterial blood of the irradiated rat at a rate approximately identical with that of the removal of cross-transfused normal leukocytes. 3. Many leukemic cells remained in the arterial blood of normal recipent rats during the 90 miniute observation time after cross-circulation. 4. It was postulated that leukocytes transfused its rats by this method remain viable. In the irradiated leukopenic recipient, the rapid arterial blood removal rate of transfused cells may represent distribution in a depleted total body leukocyte "pool." Similarly, distribution may occur more slowly in normal animals with normally saturated tissue stores of leukocytes. 5. There was no indication that a large dose of total body irradiation increased the capacity of the reticulo-endothelial system to remove transfused leukocytes from circulation.

Viability and Distribution of Leukocytes Following Cross-Circulation Experiments Between Leukemic and Normal or Irradiated Rats

Abstract 1. Recipient rats were observed following periods of circulatory mixing with normal or leukemic donor animals. 2. The normal rat tail appears to store leukocytes in its vascular channels, as evidenced by a progressively falling leukocyte count from a tail incision as the tail is "milked." 3. Comparison of irradiated rat tail and arterial blood leukocyte counts during and after cross-circulation revealed the tail values to be consistently higher than the simultaneous arterial levels. This observation excludes a major removal and destructive mechanism by some organ such as the lung as a major cause of the rapid decrease in arterial blood leukocyte levels noted in irradiated rats after the cross-circulation procedure. 4. Tail blood leukocyte counts of irradiated, cross-transfused rats remained higher than those of control irradiated rats for at least three days. 5. After cross-circulation with leukemic donor rats, leukemic cells were identified in the tail blood of normal rats for at least three days. 6. Prolonged observations revealed that 2 irradiated Sprague-Dawley strain recipients of leukemic cells from Sherman strain leukemic donors survived the severe radiation damage but developed leukemia. This observation suggests that transfused leukemic white blood cells protect against radiation damage. 7. Five normal Sherman strain rats developed leukemia after cross-circulation with leukemic Sherman donors. This incidence (50 per cent) of transmission to adult animals is much higher than has been reported by conventional methods of transfer. 8. The observations reported support the concept of viability of leukocytes transfused in rats by this method, and suggest that at least one site of noncirculating transfused leukocytes is within sluggish vascular channels.

Experiments on immunization of guineapigs with normal and leukemic human white blood cells.

Experiments on immunization of guineapigs with normal and leukemic human white blood cells.