OBJECTIVETo compare the value of fluorescence in situ hybridization(FISH) with that of immunohistochemistry in detecting ALK gene translocations and ALK fusion protein in anaplastic large cell lymphoma (ALCL) and investigate the possibility of FISH working on paraffin-embedded ALCL tissue.

METHODSDual-color FISH and ALK-1 immunohistochemistry were used to detect ALK gene translocation and ALK fusion protein in 22 paraffin-embedded ALCL cases.

RESULTSThe digestion time of tissue section was a key-point in the operating of FISH in paraffin-embedded tissue. ALK fusion protein expression was detected with ALK-1 antibody in 12 of the 20 systemic ALCL; it was not detected in 2 primary cutaneous ALCL. The Dual-color FISH results were 100% consonant with the immunohistochemical results.

CONCLUSION(1) Generally, immunohistochemical method is the first choice in detecting ALK gene translocation, but when conditions permit, FISH can be the first choice. (2) By the optimization of experimental conditions, FISH can be successfully performed on paraffin-embedded tissue.

Humans ; In Situ Hybridization, Fluorescence ; methods ; Lymphoma, Large-Cell, Anaplastic ; genetics ; pathology ; Paraffin Embedding ; methods ; Protein-Tyrosine Kinases ; genetics ; Receptor Protein-Tyrosine Kinases ; Reproducibility of Results ; Translocation, Genetic

Discoidin domain receptor 1(DDR1)is a critical member of the receptor tyrosine kinase family.It may be related to tumor invasion and metastasis,and the abnormal activation of DDR1 can lead to the occurrence and development of malignant tumors,inflammation,and fibrosis.DDR1 are involved in cell adhesion,migration,proliferation,secretion of cytokines,and remodeling of extracellular matrix,thus playing a critical role in various pathophysiological processes of the human body.In this review,we demonstrate the research progress of DDR1 in breast cancer and other malignant tumors,in order to provide a new theoretical basis for the prevention and treatment of breast cancer and other tumors.
Breast Neoplasms/genetics* ; Cell Adhesion ; Discoidin Domain Receptor 1 ; Female ; Fibrosis ; Humans ; Receptor Protein-Tyrosine Kinases/genetics*

OBJECTIVETo detect the expressions of receptor tyrosine kinases (RTKs) mRNA and protein and to explore potentially promising tumor markers and conceivable drug target in bladder cancer.

METHODSThe expressions of RTKs mRNA and protein in tissue from invasive urothelial carcinoma of the bladder were examined by real-time quantitative PCR array and cytokine antibody array, with normal bladder tissue as control. The Results were analyzed using bioinformatic approaches.

RESULTSThe expressions of TGFA, STAB1, SERPINE1, ANGPT2, SPINK5, ANGPTL1, PROK1, MDK, CXCL9, GRN, RUNX1, VEGFA, and TGFB1 were obviously upregulated in bladder cancer tissue, while those of EDIL3, PTN, CCL2, PDGFD, FGF13, KITLG, FGF2, SERPINF1, and TNF were downregulated. ALK, Btk, EphB2, ErbB4, PDGFR-α, ROS, Tie-2, Tyk2, and VEGFR3 were over-expressed in bladder cancer, while FRK, Fyn, IGF-IR, Insulin R, Itk, JAK1, JAK3, and LCK were low-expressed.

CONCLUSIONVascular endothelial growth factor/platelet-derived growth factor-targeted therapies may play an active role in treating carcinoma of bladder.

Carcinoma, Transitional Cell ; metabolism ; Humans ; RNA, Messenger ; genetics ; Receptor Protein-Tyrosine Kinases ; genetics ; metabolism ; Urinary Bladder Neoplasms ; metabolism

OBJECTIVETo investigate mutations of anaplastic lymphoma kinase (ALK) in Chinese children with neuroblastoma (NB).

METHODSGenomic DNA was extracted from 22 cases of paraffin-embedding NB tumor tissues. Gene mutations in the exons 20-26 which were mutational hotspots of ALK were analyzed by PCR-DNA direct sequencing.

RESULTSA novel synonymous mutation C3586T (Leu1196Leu) and a known synonymous mutation C3375A (Gly1125Gly) were found and located at exon 23 and exon 21 of ALK respectively. There were 10 cases (46%) of known synonymous mutation C3375A in 22 cases of NB. The C3375A allelic frequency was 27%. No statistically significant correlation was found between mutation C3375A and clinical parameters of NB such as age, sex, metastasis and tumor differentiation. Mutation was not found in the other 5 exons.

CONCLUSIONSA novel ALK gene synonymous mutation C3586T was identified using PCR-DNA sequencing. A known mutation C3375A in ALK was successfully identified in children, and its incidence is not influenced by the clinical features of childhood NB.

Child ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation ; Neuroblastoma ; genetics ; Polymerase Chain Reaction ; Receptor Protein-Tyrosine Kinases ; genetics

OBJECTIVETo correlate the abnormal expression of anapastic lymphoma kinase (ALK) protein with the genetic and epigenetic changes of ALK, and to analyze its clinical application in pediatric neuroblastoma.

METHODSThree neuroblastoma (NB) cell lines (two ALK positive: SH-SY5Y and SK-N-SH, one ALK negative: SK-N-AS) and 43 paraffin-embedded NB tissues were included in the study. In both cell lines and clinical cases, immunohistochemistry was used to detect ALK protein expression; PCR and Sanger sequencing were used to detect ALK point mutation; fluorescence in situ hybridization (FISH) was used to detect ALK abnormality and bisulfite sequencing PCR (BSP) was used to detect methylation of CpG island in the promoter area of ALK.

RESULTSThe cell lines SH-SY5Y and SK-N-SH were positive for ALK expression (cytoplasm), while the SK-N-AS was negative; among the 43 cases of NB, 26 (60.5%, 26/43) were positive for ALK protein (membrane and cytoplasm), and the rest were negative. Survival analysis showed ALK protein expression was related to survival time, with ALK positive cases having shorter survival time than ALK negative cases (P = 0.020). But ALK protein expression had no association with tumor differentiation (P = 0.503), tumor sites (P = 1.000) and age of patients (P = 0.063). FISH showed ALK amplification in two cases (4.6%, 2/43), ALK gain was found in 30 cases (69.7%, 30/43), and the remaining cases had normal ALK copy (25.6%, 11/43). The presence of extra copies (amplification and gain) of ALK was associated with ALK positive protein expression (P = 0.020), but there was no association with tumor differentiation (P = 1.000), tumor sites (P = 0.775) and age of patients (P = 0.328). No point mutation was found in all three cell lines. Of the 43 NB cases, only one case (2.3%, 1/43) showed point mutation in exon 23, and was a synonymous mutation [A1200A (G4552C)]. The case was ALK negative, but the patient died two months after diagnosis. BSP analysis showed that CpG island in ALK promoter region were all unmethylated in three cell lines and 6 NB cases (including 3 ALK positive, 3 ALK negative).

CONCLUSIONSALK protein is expressed in most NB, and the expression indicates poor outcome. ALK expression is associated with extra copies of ALK, but there is no association with the methylation status of CpG island of ALK; the presence of extra copies of ALK is the most common genetic aberration in NB. Point mutation of ALK is rare, and may predict poor prognosis in pediatric NB.

Adolescent ; Cell Line, Tumor ; Child ; Exons ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Neuroblastoma ; enzymology ; genetics ; Receptor Protein-Tyrosine Kinases ; genetics ; metabolism

The aim of study was to investigate the killing effect of double suicide gene system mediated by retroviral vector on K562 cells in vivo and ex vivo. CDglyTK gene was transfected into PA317 cells by using lipofectamine. K562 cells were infected with viral supernatant. K562/CDglyTK cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV). Mice were randomly divided into three groups: tumor formation, tumor inhibition and tumor therapy. Each mouse was implanted with K562/CDglyTK cells or K562 cells. The results indicated that the killing effect of 5-FC in combination with GCV on K562/CDglyTK was more significant than using 5-FC or GCV alone. In vivo study showed that after being injected subcutaneously with K562 cells and K562/CDglyTK cells, there was not obvious difference in tumor formation rate of mice, 5-FC + GCV could suppress tumor formation of the K562/CDglyTK cells. After being treated with 5-FC and GCV, the median tumor volume of mice implanted with K562/CDglyTK cells decreased obviously, compared with the control group. Their median survival was significantly prolonged. It is concluded that double suicide genes are more effective for killing effect on K562 cells in vivo and in ex vivo. It may be applicable to clinical gene therapy.
Cytosine Deaminase ; genetics ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; Protein-Tyrosine Kinases ; genetics ; Receptor Protein-Tyrosine Kinases ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; genetics ; Recombination, Genetic ; Retroviridae ; genetics

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic tumor originated from hematopoietic stem cells. FLT3 is an important receptor tyrosine kinase in cell signal transduction pathway and one of the common mutated genes in AML. AML patients with FLT3-ITD mutation have a poor prognosis and tendency to relapse. Therefore, early identification of FLT3 gene mutation and selection of appropriate treatment are particularly important. Currently, the small moleculetargeted drugs have been new treatment methods for AML patients with FLT3-ITD mutation, but accompanied drug resistance need to be solved. This paper reviews the mechanism of FLT3 mutation, the clinical significance of FLT3 mutation in AML, FLT3 inhibitors and drug resistance mechanism.
Humans ; Mutation ; Protein Kinase Inhibitors/therapeutic use* ; Signal Transduction ; Receptor Protein-Tyrosine Kinases/therapeutic use* ; Leukemia, Myeloid, Acute/drug therapy* ; fms-Like Tyrosine Kinase 3/genetics*

Mer is one member of Axl receptor tyrosine kinase family, and its ligand Gas6 can stimulate activity of Mer receptor tyrosine kinase after binding it, and then activate the downstream signal transduction pathway, Mer participates in cell inflammation, apoptosis, tumorigenesis, thrombosis and hemostasis. Rencet advances of study on Mer function were reviewed, and its potential prospects of antithrombosis and antitumor were discussed in this article.
Animals ; Fibrinolytic Agents ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Receptor Protein-Tyrosine Kinases ; genetics ; metabolism ; Signal Transduction ; c-Mer Tyrosine Kinase

OBJECTIVEFMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is constitutively activated in (70-90)% pediatric patients with acute myeloid leukemia (AML) and appears to confer an adverse prognosis. Although several FLT3-selective small molecule inhibitors and antibodies were developed with varied degrees of success, to address the specificity and resistance, new approaches for specifically targeted FLT3 are needed and RNA interference is a promising choice. The aim of the present study was to investigate the efficacy of suppression of FLT3 induced by small hairpin interfering RNA (shRNA) on myeloproliferation and apoptosis in an acute monocytic leukemia (AMOL) cell line THP-1.

METHODSFLT3-targeted small hairpin interfering RNA (FLT3-shRNA) was designed and synthesized by transcription system in vitro was transfected into THP-1 cells. Firstly FLT3 mRNA level was detected by semi-quantitative RT-PCR and FLT3 protein level was detected by flow cytometry (FCM) to verify the efficacy on FLT3-shRNA interference at 48 h after transfection. Cell growth viability was measured at 24 h, 48 h and 72 h after treatment with CCK-8. The distribution of cell cycle was assayed by FCM, and apoptosis was analyzed by DNA Ladder and Annexin V-FITC Staining at 48 h.

RESULTSFLT3 targeted shRNAs was synthesized successfully and the concentration of 15 nmol/L for 48 h could obtain desirable downregulation of FLT3 expression, the inhibitory percentages of FLT3 mRNA and protein were (72.95 +/- 2.07)% and (65.39 +/- 5.57)%, respectively. The suppression of FLT3 induced by FLT3-shRNA resulted in marked inhibition of cell growth and the inhibitory percentages were (36.66 +/- 3.67)% at 48 h, (35.56 +/- 0.73)% at 72 h. FLT3-shRNA induced the inhibition of cell cycle from G(0)/G(1) phase to S phase, the percentage of sub-G(0)/G(1) phase (65.71 +/- 4.47)% was higher than those in the PBS-control group (52.23 +/- 2.98)%, NC-shRNA control group (51.81 +/- 1.44)%, P < 0.01; the percentage of S phase (25.11 +/- 2.70)% was lower than those in the PBS-control group (34.41 +/- 4.07)% and NC-shRNA control group (32.50 +/- 1.46)%, P < 0.05. Furthermore treatment with FLT3-shRNA for 48 h resulted in clear apoptosis ladder, the percentage of early apoptosis detected by Annexin V-FITC was (18.59 +/- 2.07)% which was significantly higher than that in the PBS-control group (4.00 +/- 0.50)% and the NC-shRNA control group (6.06 +/- 0.70)%, P < 0.001.

CONCLUSIONThe suppression of FLT3 induced by the shRNA can effectively inhibit cell proliferation, and apoptosis induction on THP-1 cells, which indicates that this approach may bear the therapeutic potential on childhood AMOL.

Apoptosis ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Child ; Humans ; Leukemia, Monocytic, Acute ; enzymology ; pathology ; Protein-Tyrosine Kinases ; metabolism ; RNA Interference ; physiology ; RNA, Small Interfering ; pharmacology ; Receptor Protein-Tyrosine Kinases ; metabolism ; fms-Like Tyrosine Kinase 3 ; metabolism

Idiopathic myelofibrosis is a type of chronic myeloproliferative disorders characterized by splenomegaly, a leukoerythroblastic blood picture, teardrop poikilocytosis, in various degrees of bone marrow fibrosis and extramedullary hematopoiesis. In this paper, the biological characters and pathogenesis of idiopathic myelofibrosis such as mutation of tyrosine kinase receptor, mutation of GABA transporter 1, JAK2 mutation and c-MPl mutation, as well as other pathogenesis related with idiopathic myelofibrosis were reviewed.
GABA Plasma Membrane Transport Proteins ; genetics ; Humans ; Janus Kinase 2 ; genetics ; Mutation ; Primary Myelofibrosis ; etiology ; genetics ; Receptor Protein-Tyrosine Kinases ; genetics ; Receptors, Thrombopoietin ; genetics