tretinoin and dimethyl-sulfate

Induction of differentiation of HL-60 human myeloid cells profoundly affected expression of calreticulin, a Ca(2+)-binding endoplasmic reticulum chaperone. Induction with Me(2)SO or retinoic acid reduced levels of calreticulin protein by approximately 60% within 4 days. Pulse-chase studies indicated that labeled calreticulin decayed at similar rates in differentiated and undifferentiated cells (t(12) approximately 4.6 days), but the biosynthetic rate was <10% of control after 4 days. Differentiation also induced a rapid decline in calreticulin mRNA levels (90% reduction after 1 day) without a decrease in transcript stability (t(12) approximately 5 h). Nuclear run-on analysis demonstrated rapid down-regulation of gene transcription (21% of control at 2 h). Differentiation also greatly reduced the Ca(2+) content of the cells (25% of control), although residual Ca(2+) pools remained sensitive to thapsigargin, ionomycin, and inositol trisphosphate. Progressive decreases were also observed in levels of calnexin and ERp57, whereas BiP/GRP78 and protein disulfide isomerase were only modestly affected. Ultrastructural studies showed a substantial reduction in endoplasmic reticulum content of the cells. Thus, terminal differentiation of myeloid cells was associated with decreased endoplasmic reticulum content, selective reductions in molecular chaperones, and diminished intracellular Ca(2+) stores, perhaps reflecting an endoplasmic reticulum remodeling program as a prominent feature of granulocytic differentiation.

Topics: Calcium-Binding Proteins; Calreticulin; Cell Differentiation; Cell Line; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Kinetics; Molecular Chaperones; Ribonucleoproteins; RNA, Messenger; Sulfuric Acid Esters; Time Factors; Transcription, Genetic; Tretinoin

Three types of peptidylarginine deiminase (PAD), which converts a protein arginine residue to a citrulline residue, are widely distributed in animal tissues. Little is known about PAD of hemopoietic cells. We found that PAD activity in human myeloid leukemia HL-60 cells was induced with the granulocyte-inducing agents retinoic acid and dimethyl sulfoxide and with the monocyte-inducing agent 1alpha,25-dihydroxyvitamin D(3). We cloned and characterized a PAD cDNA from retinoic acid-induced cells. The cDNA was 2,238 base pairs long and encoded a 663-amino acid polypeptide. The HL-60 PAD had 50-55% amino acid sequence identities with the three known enzymes and 73% identity with the recently cloned keratinocyte PAD. The recombinant enzyme differs in kinetic properties from the known enzymes. Immunoblotting and Northern blotting with an antiserum against the enzyme and the cDNA, respectively, showed that a protein of approximately 67 kDa increased concomitantly with increase of mRNA of approximately 2.6 kilobases during granulocyte differentiation. During monocyte differentiation the same mRNA and protein increased as in granulocyte differentiation. Neither the enzyme activity nor the protein was found in macrophage-induced cells. These results suggested that expression of the PAD gene is tightly linked to myeloid differentiation.

Topics: Amino Acid Sequence; Base Sequence; Calcitriol; Cell Differentiation; DNA Primers; Enzyme Induction; Gene Library; Granulocytes; HL-60 Cells; Humans; Hydrolases; Keratinocytes; Kinetics; Molecular Sequence Data; Protein-Arginine Deiminase Type 4; Protein-Arginine Deiminases; Recombinant Fusion Proteins; Sequence Alignment; Sulfuric Acid Esters; Tetradecanoylphorbol Acetate; Tretinoin