Svend Peter Klinken

A novel ADP-ribosylation like factor (ARL-6), interacts with the protein-conducting channel SEC61β subunit

We report here the isolation of a new member of the ADP‐ribosylation factor (ARF)‐like family (ARL‐6) present in the J2E erythroleukemic cell line, but not its myeloid variants. Consistent with this lineage‐restricted expression, ARL‐6 mRNA increased with erythropoietin‐induced maturation of J2E cells, and decreased with interleukin 6‐induced differentiation of M1 monoblastoid cells. In tissues, ARL‐6 mRNA was most abundant in brain and kidney. While ARL‐6 protein was predominantly cytosolic, its membrane association increased following exposure to GTP‐γS, like many members of the ARF/ARL family. Using the yeast two‐hybrid system, six molecules which interact with ARL‐6 were identified including SEC61β, a subunit of the heterotrimeric protein conducting channel SEC61p. Co‐immunoprecipitation of ARL‐6 confirmed a stable association between ARL‐6 and SEC61β in COS cells. These results demonstrate that ARL‐6, a novel member of the ADP‐ribosylation factor‐like family, interacts with the SEC61β subunit.

Myeloid Leukemia Factor 1 inhibits erythropoietin-induced differentiation, cell cycle exit and p27Kip1 accumulation

Myeloid leukemia factor 1 (MLF1) is a novel oncoprotein involved in translocations associated with acute myeloid leukemia (AML), especially erythroleukemias. In this study, we demonstrate that ectopic expression of Mlf1 prevented J2E erythroleukemic cells from undergoing biological and morphological maturation in response to erythropoietin (Epo). We show that Mlf1 inhibited Epo-induced cell cycle exit and suppressed a rise in the cell cycle inhibitor p27Kip1. Unlike differentiating J2E cells, Mlf1-expressing cells did not downregulate Cul1 and Skp2, components of the ubiquitin E3 ligase complex SCFSkp2 involved in the proteasomal degradation of p27Kip1. In contrast, Mlf1 did not interfere with increases in p27Kip1 and terminal differentiation initiated by thyroid hormone withdrawal from erythroid cells, or cytokine-stimulated maturation of myeloid cells. These data demonstrate that Mlf1 interferes with an Epo-responsive pathway involving p27Kip1 accumulation, which inhibits cell cycle arrest essential for erythroid terminal differentiation.

Diaminofluorene is more sensitive than benzidine for detecting hemoglobin in erythropoietin responsive J2e cells

We have used the diaminofluorene stain to detect hemoglobin production in J2E cells following erythropoietin-induced differentiation. The pseudo-peroxidase activity of hemoglobin produces a colored product, fluorene blue, which can be measured spectrophotometrically. We found that the absorbance varied with time and concentration of hemoglobin, making it unsuitable for rapid, routine use. However, hemoglobin content could be determined from the initial reaction rate and this correlated extremely well with the number of benzidine positive cells. When used as a direct cytochemical stain diaminofluorene was shown to be more sensitive than benzidine in detecting hemoglobin-producing J2E cells.

Identification of the enzymic control point in ‘de-differentiation’ of oestrogen synthesis in superovulated rat ovary

The superovulated rat model was used to investigate the enzymic focus for the decrease in oestrogen synthesis which occurs in ovary at the time of ovulation. Radioimmunoassays of progesterone, 17 alpha-hydroxyprogesterone, androstenedione, testosterone and 17 beta-oestradiol were used to measure the steroid concentrations in plasma for 6 days after the initiation of follicular development with pregnant mares gonadotropin, and the long-term and acute effects of choriogonadotropin on these circulatory concentrations. The results showed that the cross-over point following the mid-cycle administration of gonadotropin was between 17 alpha-hydroxyprogesterone and androstenedione, and suggested that choriogonadotropin affected the 17 alpha-hydroxyprogesterone 17:20 lyase. In vitro assay of this microsomal enzyme confirmed that choriogonadotropin given in vivo at intervals before death caused 50% reduction in 17:20 lyase activity in 4 h and 93% reduction in 6 h. It was concluded that the synthesis of oestrogens declined following ovulation because the substrate (testosterone) was not available in sufficient concentration for the aromatase enzymes to use it.