Erythroid differentiation denucleation factors (EDDFs) function as intrinsic, post-erythropoietin regulators for mammalian erythroid terminal differentiation.

Regulatory factors other than erythropoietin (Epo) dependence, that control mammalian erythroid terminal differentiation, are currently uncertain. Here we report the existence of erythroid differentiation factors in erythroid cytoplasm. Purification of these factors from cultured Friend virus anaemia (FVA)-infected mouse splenic erythroblasts was carried out using isoelectrophoresis and high performance of liquid chromatography techniques. We have identified intracellular erythroid differentiation denucleation factors (EDDFs) that were able to mediate the events of post-Epo-dependent erythroblast terminal differentiation. Purified EDDF proteins bound specifically to the enhancer HS2 sequence of the globin gene activated the expression of haemoglobin in mouse erythroleukaemia and K562 erythroleukaemic cells and promoted them to differentiate into mature erythrocytes. EDDF proteins began to emerge at the pro-early erythroblast stages upon exposure to Epo in culture, and increased dramatically in early erythroblast stage. The dynamic of EDDF expression and its action on the key events of erythroblast differentiation and denucleation appeared to be closely consistent with its spatiotemporal distribution. These results suggest that EDDFs are the critical intracellular regulatory factors that may act as the successive regulators to Epo, responsible for the final stages of erythroid terminal differentiation.

[Differentiation and malignant suppression induced by mouse erythroid differentiation and denucleation factor on mouse erythroleukemia cells].

OBJECTIVE: To investigate the roles of mouse erythroid differentiation and denucleation factor (MEDDF), newly cloned in our laboratory, in erythroid terminal differentiation. METHODS: Mouse erythroleukemia cells (MEL) were transfected with eukaryotic expression plasmid pcDNA-MEDDF. The changes of cell growth rate, mitotic index and colony-forming rate in semi-solid medium were investigated. The expressions of c-myc and beta-globin genes were analysed by semi-quantitative RT-PCR. RESULTS: MEL cells transfected with pcDNA-MEDDF showed significant lower growth rate, mitotic index, and colony-forming rate in semisolid 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 was decreased by 66.3%. CONCLUSIONS: MEDDF can induce differentiation of MEL cell, and suppress its malignancy likely.

The role of cytoskeletal elements in the two-phase denucleation process of mammalian erythroblasts in vitro observed by laser confocal scanning microscope.

The cytoskeletal elements in the denucleation processes were observed using immunofluorescence and laser confocal scanning microscopy in the Friend virus (FVA) infected splenic erythroblasts of BALB/c mice. When cultured in the presence of erythropoietin (EPO), it was shown that the synchronized erythroid precursor cells proceeded to an autonomous nuclear extrusion when the three types of cytoskeletal elements were observed contributing to different phases of that process. The vimentin intermediate filament (IF) was shown as the nuclear anchorage elements with binding sites anchored from the nuclear lamina to the center as well as to the plasma membrane periphery. A dense perinuclear layer of vimentin fluorescence in erythroblasts was observable during the periods of 12, 24 and 36 hrs. in vitro culture. The amount of vimentin IF per cell was higher than that of tubulin and F-actin at 12-24 hrs. culture, but the vimentin filaments were observed to brake down and decreased steadily when the cells became differentiated into late erythroblasts at 36-48 hrs. Such an attenuation of vimentin filaments may facilitate the eccentric movement of the nucleus which can be regarded as the initial step (phase) of denucleation. The fluorescent intensity of tubulin and actin exhibited a significant rise and aggregated between the extruding nucleus and the incipient reticulocyte prior to and during the processes of denucleation, what indicated that the actin filaments and microtubules may play roles in the second phase of the denucleation process, or final commitment of enucleation. The erythroid differentiation-denucleation factor (EDDF), as an intrinsic factor, involved in the denucleation events, was also discussed.

[The antifertility effect of gossypol plus testosterone and estrogen].

In order to reduce the side effect of gossypol, gossypol was used in combination with steroid hormones so that the dose of both drugs can be reduced. Silastic capsules containing testosterone (T) + estradiol (E) were implanted under the skin male wister rats for 8 weeks. After removing the implants, testosterone was given orally at the dose of 15 mg.kg-1 which is only 50% of the usual antifertility dose. Mating tests showed that the male rats became infertile. Microscopic examination of the heart, liver and kidneys showed no pathologic changes. The treated rats gained body weight as well as the controls. The fertility of the treated rats recovered four to five weeks after treatment. Thus, gossypol in combination with testosterone and estrogen exhibited a low degree of side effect and high antifertility activity.

[The study of the relationships between the processes of denucleation and vimentin, lamin].

By employing K-RRneo cells which were cybridized between rabbit reticulocytes and the K562 cells, the relationships between the denucleation and vimentin/lamin were studied. It was demonstrated that, accompanying with the differentiation and denucleation of K-RRneo cells, the expression of vimentin mRNA and lamin proteins were significantly reduced. These results testified our earlier conclusion drawn from the whole mount and Western blot studies, and would provide a basis for further elucidation of the mechanism of erythroblast denucleation.

[Nuclear matrix-intermediate filament scaffold of cybrid cells crossed between rabbit reticulocytes and K562 cells].

To elucidate the role of the mammalian erythroid cytoplasmic factor on erythroid cell denucleation, cybridization of neo gene transferred rabbit reticulocytes with human K562 erythroleukemia cells was performed, the structure and composition of the nuclear matrix-intermediate filaments (NM-IF) system of the reticulocytes, K562 cells and the cybrids between them, the K-RRneo Cells, were studied by employing the techniques of selective extraction and whole mount electron microscopy. It was shown that regional condensation of nuclear matrix with a thinned nuclear lamina were seen in the cybrids, while the intermediate filaments exhibited a network pattern reorganization comparatively similar with those of reticulocytes but different from the radiation pattern of the K562 cells. SDS-PAGE and western blot analysis also revealed that the proteins affinity to vimentin antibody in the cybrids migrate in a pattern similar with that of the rabbit reticulocytes, both of them with only residual depolymerized vimentin complex but not the polymerized vimentin band of 55 kd. These results suggested that the factors in the cytoplasm of the rabbit reticulocytes might function through a mechanism of vimentin depolymerization or blockage of gene expression, which finally resulted in destruction of intermediate filaments that facilitates the expellation of nucleus from cytoplasm.

Studies on the cell phenotype characteristics of hybrid cells crossed between rat nucleated erythroblasts and mouse plasmocytoma (SP2/O) cell line.

The present study is designed to investigate the regulatory effect of mammalian erythroblasts, prior to naturally-occurring denucleation, on malignancy of mouse plasmocytoma cells and the possibility of reactivation of the pyknotic late erythroblast nuclei in hybrid cells crossed between rat intermediate or late erythroblasts of 15-day Wistar rat embryonic livers, and mouse plasmocytoma (SP2/O) cell lines. Results indicated that: (i) Suppression of tumorigenicity and reversion of the malignant phenotype were observed in hybrid cells in a similar way as those of cybrid cells crossed between reticulocytes and myeloma cells as we reported previously, thus providing further evidence to support the hypothesis that some regulatory substances already existed in mammalian intermediate and late erythroblasts long before nuclear extrusion. (ii) Appearance of positive histochemical reaction for hemoglobins in cytoplasm and electrophoretic bands of rat and mouse globin chains in hybrid cell lysate were identified. The transcripts of mouse globin genes could be readily detected by nucleic acid hybridization technique with mouse beta-globin gene probes. (iii) Reassuming of rat chromosome and globin gene products synthesis in hybrid cell indicates that the originally pyknotic nuclei of late erythroblasts could be reactivated to assume functional activity after cell hybridization. The mechanism of regulatory effect and its possible relation to naturally occurring denucleation in developing mammalian red blood cells were discussed.

[Isolation of polynectin and its comparison with fibronectin].

A gelatin-binding protein polynectin (PN) was isolated from porcine plasma by affinity chromatography in sequence of columns of Sepharose 4B coupled with gelatin, arginine and heparin and then by gel filtration through Sephadex G-200. This protein was bound strongly to arginine column and thus could be separated from fibronectin (FN). PN is similar to FN with respect to bind characteristics to affinity gels and with a molecular weight of about 450,000 in PAGE, PN being composed of several small subunits of the same molecular weight linked by disulfide bonds. Results of immunodiffusion and ELISA shows that: no immune reaction was observed between anti-PN antibody and FN and PN existed only in porcine plasma. but not in other animal (bovine, goat, mouse and rat) or human plasma. PN exhibited no cell-attachment-promoting effects on CHO cell. These indicate that PN was a glycoprotein different from FN. Moreover, a high titer (1:32) rabbit antiserum against porcine plasma FN (free from PN) was prepared from immunized rabbit, from which affinity purified anti-FN antibody was obtained through FN-Sepharose 4B column.

[Isolation and characterization of fibronectin from porcine plasma].

Electrophoretic purified fibronectin (FN) was isolated with high concentration (0.5 mg/ml plasma) from porcine plasma by affinity chromatography with gelatin-sepharose 4B and heparin-sepharose 4B. The isolated FN shows one single band (MW 450,000) both in agarose gel and PAGE, and two similar bands (MW 230,000) in the presence of 1% beta-mercaptoethanol in SDS-PAGE. The FN isolated remains its immunological and biological properties, being reactable with antibodies against human and bovine FN with strong promotion effect on CHO cell adhesion in serum free medium. These data indicate that porcine plasma is a good resource for isolation of FN.

Effect of rhodamine 123 and total glycosides from tripterygium wilfordii (GTW) on ultrastructures, survival and fertilizing capacity of human sperm in vitro.

Human sperms were treated with rhodamine 123 or GTW in vitro. Comparisons were made of survival, fertilizing capacity and ultrastructures of sperms which had been exposed to different concentrations and treatment durations of the agents. The results showed that no prominent change could be observed when treated with lower doses. However, at higher concentrations of rhodamine 123, mitochondria were found to be swollen, vacuolated and deranged instead of surrounding the flagellar axoneme in the normal spiral way, and the mitochondrial cristae were broken or had disappeared. In some sperms, cracks at the flagellar axoneme and concave defects in the acrosome were seen. The sperms lost their fertilizing capacity, though some of them were still alive, whereas the sperms treated with GTW showed no morphological damage and maintained their ability to penetrate the hamster egg. Our results suggest that rhodamine 123 is a prospect in the search for an anti-sperm contraceptive.

Effects of gossypol on Na+, K+ transmembrane fluxes in human red cells.

The effects of gossypol-acetic acid (a male contraceptive) on transmembrane fluxes of sodium and potassium in human red cells were studied, in comparison with the effects of ouabain (an inhibitor of Na+-K+-ATPase) and furosemide (an inhibitor of Na+, K+ cotransport). It was found that gossypol may be an inhibitor of outward (Na+, K+) cotransport in human red cells but has no significant effect on (Na+, K+)-ATPase. The results provide new evidence for elucidating the mechanism of hypokalemia caused by gossypol.

Studies on the regulation of malignant phenotype and gene expression in human promyelocytic leukemia cell mutant (HL-60-AR).

The present study has investigated the regulation of malignant phenotype and gene expression in a human promyelocytic leukemia cell mutant (HL-60-AR) by means of cellular engineering technique of cybridization between the fusions of the mutant cells with enucleated mouse reticulocytes. Results indicate that the cybrid cells (HL-R) incorporated with reticulocyte cytoplasts become differentiated, and the malignancy is obviously suppressed or reversed to a certain degree when compared with those of parental tumor cells. They lose the growth ability to form colony soft agar medium, become non-tumorigenic under heterotransplantation to nude mice, and are accompanied by decrease in growth rate, cellular mitotic index and DNA synthesis. No gene transcripts or homologous sequence of mRNA corresponding to c-myc oncogene can be detected in cybrid cells by Northern blot technique with cloned c-myc gene probe, suggesting that the expression of originally active c-myc has been inhibited. On the other hand, analysis, by the same molecular hybridization technique with human globin gene probe and by PAGE method, of the expression of globin gene products in cybrid cells detected at the transcription (globin mRNA) and translation (hemoglobin) levels demonstrates consistently that originally inactive human globin gene has been activated to express in passages. These results suggest that some regulatory factors existing in reticulocyte cytoplasm can regulate gene expression and reverse malignant phenotype of leukemia tumor cells.

Sites of action of gossypol studied by autoradiography and enzyme histochemistry.

The distribution of 14C-gossypol acetate was studied by autoradiography in male rats after intraperitoneal or intratesticular injection. Accumulation of radioactivity was found in testis, kidney and liver, while there was little in brain, pituitary and epididymis. In testis, high accumulation occurred in interstitial cells, with low levels in Sertoli cells, spermatogonia and spermatocytes. In addition, the chronic effect of gossypol was assessed by enzyme histochemistry with thiamine pyrophosphate, alpha-glycerophosphate dehydrogenase, and by lipid stain. In the treated animals an increased number of luminal exfoliated cells (Sertoli cells, germ cells and spermatids) was noted, which showed positive reactions. The results suggest both direct and indirect effects of gossypol on testicular functions.

Evaluation of the renal tubulo-toxic effect of gossypol by urinary protein analysis with SDS-polyacrylamide gel electrophoresis.

The present study assessed the urinary protein with SDS-PAGE technique and by using gentamicin as a positive control for a comparative study to evaluate the renal toxic effect of gossypol. Results indicated that gentamicin could induce proteinuria by alteration of glomerular permeability to cause over-filtration of high molecular weight proteins, damage the brush border (BB) membrane of proximal renal tubules and impair tubular reabsorption function. Gossypol could also induce proteinuria in certain cases of guinea pigs but not at all in gossypol-treated rats. However, an increase in low molecular weight protein bands (M.W. range 20-30 kd) in urinary electrophoregram of gossypol-treated rats were detectable.