OBJECTIVETo investigate the roles of mouse erythroid differentiation and denucleation factor (MEDDF), a novel factor cloned in our laboratory recently, in erythroid terminal differentiation.

METHODSMouse erythroleukemia (MEL) cells were transfected with eukaryotic expression plasmid pcDNA-MEDDF. Then we investigated the changes on characteristics of cell growth by analyzing cells growth rate, mitotic index and colony-forming rate in semi-solid medium. The expressions of c-myc and beta-globin genes were analysed by semi-quantitative RT-PCR.

RESULTSMEL cells transfected with pcDNA-MEDDF showed significant lower growth rate, mitotic index, and colony-forming rate in semi-solid medium (P<0.01). The percentage of benzidine-positive cells was 32.8% after transfection. The expression of beta-globin in cells transfected with pcDNA-MEDDF was 3.43 times higher than that of control (MEL transfected with blank vector, pcDNA3.1), and the expression of c-myc decreased by 66.3%.

CONCLUSIONSMEDDF can induce differentiation of MEL cell and suppress its malignancy.

Activins ; genetics ; pharmacology ; Animals ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; Friend murine leukemia virus ; Globins ; biosynthesis ; genetics ; Inhibin-beta Subunits ; genetics ; pharmacology ; Leukemia, Erythroblastic, Acute ; metabolism ; pathology ; Mice ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; Transfection ; Tumor Cells, Cultured

Gossypol has been shown to cause a side effect hypokalemia. This study was designed to evalulated the possible role of gossypol in relation to renal potassium loss by using histochemical method for comparative analysis of gossypol and gentamicin, a well known tubulo-toxic agent, as positive control with respect to nephrotoxic demage in rats and guinea pigs. The results indicated that gossypol had obvious effect on reabsorption function by inducing transitional decrease of membrane enzymes activities of renal tubular cells but had no effect as comparable with gentamicin to cause structureal damage of tubular cells.

The present study was designed to investigate the effect of gossypol on the reabsorption and excretion of ~(42)K in rats and guinea pigs by using autoradiographic technique, and selected the well known tubulo-toxic agent, gentamicin as a positive control for a comparative study to evaluate whether gossypol exerts nephrotoxic effect. Our results confirmed that gentamicin could induce significant decrease or inhibit ~(42)K reabsorption and cause structural damage of renal tubules. Gossypol could also affect the reabsorption function of proximal tubule, but did not appear to act as a tubulotoxic agent comparable with gentamicin to cause injury of the renal tubules.

Junctions of myocardial cell, specially the intraeellular junctions of transverse tubule system with sarcoplasmie reticulum membranes were observed and described in the present paper. The tubular invaginations of the sarcolemma constitutes the transverse tubule (T tubule) which surround the myofibril in rabbit. The transverse tubular system of cardiac muscle are well developed. The tubule has an elliptical shape in cross section and 15 nm in diameter and is constantly located at the level of Z line. The sarcoplasmic retieulum consists of a simple plexiform arrangement of tubular elements forming a loose network around the myofibrillae. Small terminal expansions of the reticulum are closely applied to the membrane of the T tubules to form Diad. The membranes of the small flattened expansions of the reticulum and T tubules are in contact with each other, but the lumina of the two elements do not communicate. There is a gap of about I0 nm between them and foot processes from the membrane of sarcoplasmic reticulum are regularly extended toward the Ttubules. In addition, intracellular membranous junctions are observed between T tubules and sarcoplasmic reticulum of Diad. Each junction with membrane area of about 375 nm long becomes thicker and divides into two layers. Between them there is a gap of 31 nm wide, in which it is filled up with electron, dense material, forming some discontinuous spots which consisted of dense and light areas arranged at regular intervals.

A method described by Harrison has been adopted and modified by us for the separation of intermediate and late erythroblast cells from 15 day embryonic liver of pregnant Wistar rat. The method consisted briefly of preparation of fetal liver cell suspension and the separation of cell types in a 40% and 70% nonlinear Percoll gradient system. Using this method, we can obtain about 96% of hemogenous population of intact and viable intermediate and late erythroblasts. Examination of tho separated cells by Giemsa and benzidine staining and by electron microscopic observation indicated that no granulocytes or other white blood cells could be detected, except for some contamination of about 1% of proerythroblasts and 3% of reticulocytes in the fraction. Trypan blue vital staining demonstrated that there were over 95％ of the cells maintained viable after separation, they could be used directly for the study of cell differentiation as well as biochemical analysis. SO, this is an economical, simple and easy technique to operate which proved to be a useful mean for obtaining enrich population of intermediate and late erythroblasts in research in the field of cell biology.

Our previous study showed that malignant phenotype of human promyelocytic leukemia cells could he suppressed by fusion of them with mouse reticulocytes. In order to investigate the morphological changes for malignancy reversion, the present experiment was designed to study the microscepic and submicroscopic structure of cybrid cells and compared with their parental tumor cells. The results indicated that during the short period of cybrid cell cultivation, nuclei of numerous cybrid cells became pyknotic and eccentric, and some cells showed the process of nuclear expulsion (denucleation). The cybrids which cultivated for long period in vitro developed into more mature cells along both myeloid and erythroid differentiation pathway. The effects of mouse reticulocyte cytoplasmic factor on differentiation pathway of human promyelocytic leukemia cells were discussed.

The present study reported the effect of two male antifertility agents gossypol acetic acid and GTW on DNA of C3H10T1/2 mouse fibroblasts. Our results showed that the cells treated with gossypol or GTW at high concentration (2-3 ?g/ml) for 4 hours, show silver grains in their nuclei as much as the positive control group, N-methyl N′-nitro-N-nitrosoguanidine (MNNG) a known carcinogen. However, if the agents were used at moderate concentrations (0.5-1?g/ml), the silver grains were much less, if the concentrations of gossypol or GTW were of 0.1-0.3 ?g/ml, the silver grains were as less as the control group. In a colony-forming test, we found that the cells lost their proliferate ability, since no colonies could be formed, if gossypol or GTW were of high concentration; while at moderate or low concentrations, the colony-forming rate was as high as 8.1-10.5％. Taking all of these results into consideration, we suggest that high concentrations of gossypol or GTW can damage cell DNA severely, moderate concentration of the agents break cell DNA to a certain extent, but the cells can repair, while low concentration of gossypol or GTW exert no obvious effect on cells. The significance of these observations was briefly discussed.

The heterospecies hybrid cells(HL-N)from the fusion of human promyelocy-tic leukemia mutant cells(HL-60-AR)and mouse bone marrow nucleated red cellswere established in HAT selective medium.Malignant phenotype comparative analy-sis between parental tumor cells and hybrid cells showed that growth ability ofhybrid cells was decreased.The hybrid cells reduced their DNA synthesis rate andlost the ability of colony-forming in 0.3% soft agar medium.The cells lost tumor-producing ability when they were transplanted into nude mice also.Inhibition orreduction of c-myc oncogene expression was demonstrated by Northern molecularhybridization techniques.The ultrastructure of hybrid cells were also different fromtheir parental cells.These results mentioned above showed that the mouse bone mar-row nucleated red cells might provide some peculiar factors(both nuclear factorsand cytoplasmic factors)to inhibit the expression of HL-60-AR cell malignant phe-notypes.

The present study is designed to establish a xenograft model of human promyelocytic leukemia cell mutant (HL-60-AR) deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT) in nude mice. A solid leukemia sarcoma developed after subcutaneous inoculation with HL-60-AR cells. Comparative studies of HL-60-AR/Nu tumor cells in nude mice and cultured HL-60-AR cells in vitro revealed virtual identity as shown by light microscopic morphology, ultrastructure of cell, cytochemistry, chromosome analysis, LDH isoenzyme pattern, genetic markers and differentiated characters assay. Up to now, twelve generations haw been transmitted in rive by inoculating with the solid tumor Cells developed in nude mice. This nude mice model in which human leukemia cells grew could be considered as a useful model for in rive studies of human leukemic cells proliferation, differentiation and the screening for anti-leukemia drugs.

The experiment was designed to compare the changes of cytoskeleton (including tubulin and actin) and cell surface fibronectin (FN) between NIH 3T3 cell line and transformed NIH 3T3 cell line by genome DNA of human lung adenocarcinoma cell line AGZY. We stained and observed these cells using immunohistochemical methods with antibody against bovine brain tubulin, phalloidin and self-made affinity column purified antibody against porcine plasma FN.Our results showed that the bundles of actin and tubulin are damaged seriously, demonofrating an unclear cytoskeleton structure and diffused fluorescence over the cells when they were transformed. The amount of membrane FN on transformed cell surface decreases significantly which is only 1/9 of NIH 3T3. The FN distribution altered markedly from thin threadlike network to spots and speckles.These results suggested that cell transformation was a complex event including the changes of cytoskeleton system and cell surface glycoproteins. In addition, it might also indicate that cause and effect relationship existed between these changes and the alteration of cell phenotype and loss of growth control.