Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97

AIM: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied. RESULTS: Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10). CONCLUSION: Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.

Bcl-1 Rearrangement and Cyclin D1 Protein Expression in Multiple Myeloma Precursor Cells

The rearrangement of Bcl-1 gene (Bcl-1/IgH rearrangement) and expression of cyclin D1 in multiple myeloma (MM) precursor cells were studied and the role of cyclin D1 in the pathogenesis of MM was investigated. The BCL-1 rearrangement and cyclin D1 protein expression in 15 cases of MM were detected. By using hemi-nested polymerase chain reaction (PCR) the genomic DNA from fresh peripheral blood and bone marrow was amplified and the expression of cyclin D1 in the smears was detected by using immunohistochemical method. Ten volunteer with normal bone marrow served as control group. The results showed Bcl-1 rearrangement was detectable in 3/15 (20 %) MM patients and cyclin D, expression in 4/15 (27 % ) MM patients. BeLl-1 rearrangement and cyclin D1 protein expression were also detected in MM precursor cells. No overexpression of cyclin D1 or the rearrangement of the BeL-1 gene was found in the 10 volunteers. It was concluded that Bel-1 rearrangement and cyclin D1 protein overexpression were detected in MM precursor cells, speculating that overexpression of cyclin D1 protein may play an initial (critical) role in the pathogenesis of MM.

ESTABLISHMENT OF CELL CLONE OF LYMPHOMA AND A CELL LINE INFECTED WITH LEUKEMIA VIRUS AND STUDY ON ITS INDUCTED DIFFERENTIATION

Since 1960, the tumor strains of L6565 viral leukemia, SRS lymphoma and L783 transplantable leukemia were established successively in our laboratory. Recently, derived from the strains of threse leukemia/lymphoma, SRS-82 cell line, SACIIB2, SACIIC3 cell clones and a cell line infected with SRS leukemia virus (SRSV/3T3) were obtained at vitro. The main results of study on the biology, virology and Its Induction of differentiation are summarily reported.

ELECTRON MICROSCOPIC OBSERVATION OF DIFFERENTIATION OF LEUKEMIC CELL CLONE AFTER CFU-MIX CULTURE

By scanning and transmission electron microscopy, leukemlc celb were obwrved after CFU-Mix culture. Even though granulocytlc growth factor, erythropoietin and lymphocytlc growth factor were added at vitor, acute leukemlc celb still showed defects In differentiation and maturation. These were characterized by abnormal colony which consisted of smooth cells, bizarre shape, nuclear-cytoplasmic asynchrony In development, and appearance of nuclear bleb. However, chronic myelogenous leukemlc celb were more nature than the acute ones, manifesting in normal colony with finger- like projections and ruffled membrane. Macrophages and eosinophils could be observed. It b suggested that there b a difference In differentiation between acute and chronic leukemia cells.

DNA Methylation Analysis of Exogenous Genes Transformed into Sheep by Different Methods

[Objective] This study aimed to investigate the methylation levels of exogenous genes and promoters and the differences of protein expression in transgenic sheep obtained by different transgenic technologies. [Method] Exogenous genes eGFP （enhanced green fluorescent protein） and FGF5 （fibroblast growth factor 5） were separately transformed into sheep by somatic cell cloning, stem cell cloning and perivitelline injection to obtain transgenic sheep, with CMV as the promoter. Bisulfite sequencing method was adopted to detect the methylation status of the promoter region and coding region of exogenous genes in tail tissues of transgenic sheep. Western blot was adopted to detect the expression level of exogenous genes. [Result] The methylation level of the promoter region with stem cell cloning was the highest, followed by somatic cell cloning, while that with perivitelline injection was the lowest; the methylation level of the eGFP coding region with perivitelline injection was the highest, followed by stem cell cloning; the methylation level of the FGF5 coding region with somatic cell cloning was higher than that with perivitelline injection. The exogenous protein expression level was negatively correlated with the methylation level of the promoter region. [Conclusion] This study indicates that different transgenic methods may influence the methylation level of exogenous genes, thus affecting exogenous gene expression.

China Succeeded in Somatic Cell Cloning

Chinese scientists have succeeded in cloning a colony of cattle from fully differentiated somatic cells. The news was announced jointly by the Chinese Academy of Sciences (CAS), National Natural Science Foundation of China (NSFC) and the government of Shandong Province at a press conference held on March 7, 2002.……

Immunophenotypic analysis of abnormal plasma cell clones in bone marrow of primary systemic light chain amyloidosis patients

Background Primary systemic light chain amyloidosis （AL） is a rare plasma cell disease,our purpose was to analyze the immunophenotypic characteristics of the plasma cells in bone marrow in AL patients,and explore whether the detection of abnormal plasma cell clones in bone marrow by flow cytometry （FCM） could be used as an important indicator of AL diagnosis.Methods Fresh bone marrow samples were collected from 51 AL,21 multiple myeloma （MM）,and 5 Waldenstr（o）m＇s macroglobulinemia （WM） patients.The immunophenotype of bone marrow cells were analyzed and compared by FCM using a panel of antibodies including CD45,CD38,CD138,CD117,CD56,and CD19.Results In AL,light chain restriction could be identified in 31 cases （60.9%）,in which the λ light chain restriction was found in 24 cases （77.4%）.In MM,κ light chain restriction was found in 13 cases （61.9%）,and λ light chain restriction in eight cases.CD45 on abnormal plasma cells was negative to weakly positive in both AL and MM,but was positive to strongly positive in WM.In the bone marrow plasma cells of the 51 AL,78.4% were CD56＋,68.6% were CD117＋,and 88.2% were CD19-.While in the 21 MM cases,66.7% were CD56＋,38.1% were CD117＋,and 90.4% were CD19-.The plasmacytoid lymphocytes in the five WM patients were CD19＋ and CD56-,CD117-.Conclusion Detection of abnormal plasma cell clones in bone marrow by FCM is valuable for the diagnosis of AL.

Childhood leukemia genetic bottleneck phenomenon related to TEL-AMLI： the postulation by a mathematical model

Childhood leukemia bottleneck phenomenon is the most mysterious corollary of the prenatal origin discovery of leukemogenic chromosome translocations. The bottleneck is evidence that leukemia initiation, by in utero acquired chromosome translocations that generate functional fusion genes, is far more common than the incidence rate of corresponding leukemia. For childhood TEL-AML1 ^＋ acute lymphoblastic leukemia （ALL） this equates to approximately 100 times. Practically this means that among a hundred children born with TEL-AML1 fusion gene, only one child will later in its life develop ALL. The key data necessary for unraveling of this mystery were discovered in 2002. It was the level of TEL-AML1^＋cells＇ frequency. The bottleneck is caused by the very low body TEL-AML1^＋cell count. Only one out of a thousand B cells carries TEL-AML1 fusion gene. TEL-AML1^＋body cell count is low because TEL-AML1 fusion is generated at cell level of 103 to 104 just during the late fetal lymphopoiesis i.e. after the 36th gestational week.