Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule.

Assay of red cell progenitors by colony formation in culture is expected to allow study of early events in erythroid differentiation in animal models and in man. In this communication, a simplification of the culture method for mouse bone marrow cells originally reported by Stephenson et al. ('71) is described. In the modified procedure, plasma clot is replaced by methyl cellulose, and scoring of erythroid colonies is done directly in the plates without staining. With additional modification the system proved applicable to the culture of erythroid colonies from human bone marrow as well. The development of both mouse and human erythroid colonies was dependent on “erythroid colony stimulating activity” (E-CSA) supplied by extracts of anemic sheep plasma, or by extracts of urine from anemic patients. Over a certain range, the number of colonies was a function of dose of E-CSA.

In addition to E-CSA, these extracts also possessed erythropoietin activity as measured in plethoric mice. The possible chemical equivalence of urinary E-CSA and erythropoietin was suggested by their similar behavior on both gel filtration and affinity chromatography on agarose-concanavalin A. The finding further suggests that the culture method might prove useful for the bioassay of erythropoietin.

Granulocyte colony stimulating activity (G-CSA) was also present in the urine extracts, as detected in cultures of mouse bone marrow. Virtually all of this activity was bound on agarose-con A, whereas only a small fraction of E-CSA was retained on this material. Agarose-con A may thus be useful for the purification of erythropoietin.

Erythroid colony formation in cultures of mouse and human bone marrow: analysis of the requirement for erythropoietin by gel filtration and affinity chromatography on agarose-concanavalin A.

Murine embryonal carcinoma cells can differentiate into a varied spectrum of cell types. We observed the abundant and precocious development of neuronlike cells when embryonal carcinoma cells of various pluripotent lines were aggregated and cultured in the presence of nontoxic concentrations of retinoic acid. Neuronlike cells were also formed in retinoic acid-treated cultures of the embryonal carcinoma line, P19, which does not differentiate into neurons in the absence of the drug. The neuronal nature of these cells was confirmed by their staining with antiserum directed against neurofilament protein in indirect immunofluorescence experiments. Retinoic acid-treated cultures also contained elevated acetylcholinesterase activity. Glial cells, identified by immunofluorescence analysis of their intermediate filaments, and a population of fibroblastlike cells were also present in retinoic acid-treated cultures of P19 cells. We did not observe embryonal carcinoma, muscle, or epithelial cells in these cultures. Neurons and glial cells appeared in cultures exposed to retinoic acid for as little as 48 h. We found no evidence for retinoic acid toxicity, suggesting that the effect of the drug was to induce the development of neurons and glia rather than to select against cells differentiating along other developmental pathways.

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Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells.

LEDER and Leder1 have reported that low concentrations of n-butyrate cause Friend erythroleukaemia cells to begin globin synthesis. Apparently n-butyrate can reverse that part of viral transformation which prevents the expression of differentiation in these cells. Prasad and Sinha2 have summarised the effects of n-butyrate on neuroblastoma, HeLa, and other cell types. They and others3–10 have seen reversible inhibition of proliferation, decrease of DNA content, morphological modifications, and increases in the production of specific enzymes, such as adenylate cyclase, alkaline phosphatase, and a sialyltransferase. The present paper describes rapid, dramatic, and reversible increases in histone acetylation in the presence of n-butyrate.

n -Butyrate causes histone modification in HeLa and Friend erythroleukaemia cells

Factors regulating macrophage production and growth. Purification and some properties of the colony stimulating factor from medium conditioned by mouse L cells.

ONLY mouse 28S ribosomal RNA (rRNA) can be detected in mouse-human hybrid cells1. Because these cells contain as many as thirty-five human chromosomes, including those believed to have ribosomal RNA genes, it has been suggested that the transcription of human ribosomal RNA genes may be repressed. Similar studies have not been performed with mouse-hamster hybrid cells because hamster and mouse 28S rRNAs have only a very small difference in their electro-phoretic mobilities2. We have now made use of the observation that free ribosomes (not engaged in translation) from hamster cells form dimers in conditions where mouse free ribosomes remain as monomers3 to determine whether mouse-hamster hybrid cells contain free ribosomes as monomers, dimers or as both.

Two types of ribosome in mouse-hamster hybrid cells.

Short-lived, small RNAs in the cytoplasm of HeLa cells

We are interested in two aspects of a given type of metastatic breast cancer: which potentially cancer-relevant genes are expressed and which factors determine invasiveness. Using reverse transcription real-time PCR, we detected gene expression of 26 matrix metalloproteinases (MMPs) in MDA-MB-231 breast cancer cells, including those of MMP-12, MMP-16 variant 2, MMP-19, MMP-20, MMP-21, MMP-23, MMP-24, MMP-25, MMP-25 variant 2, MMP-L1, MMP-26, MMP-27, and MMP-28, in contrast to the 13 MMPs detected until now in these cells. We found that MMP genes are expressed at widely different levels in these cells, over five orders of magnitude. After individual siRNA-induced depletions, we found that six additional species of cancer cell MMPs promote invasiveness in MDA-MB-231 cells: MMP-3, MMP-11, MMP-12, MMP-17, MMP-19, and MMP-23, thus raising the total to 12 endogenous MMPs which do so in these cells. The data support the conclusion that some cancer cell MMPs, although expressed at low levels, are needed for cancer trait in MDA-MB-231 cells, and that several endogenous MMPs play non-redundant roles in this process. The mRNA level of MMP-11, but not of other MMPs, rose substantially following individual siRNA-targeted depletion of cancer cell MMP-17 mRNA, while no MMP mRNA increased appreciably after degradation of other MMP mRNAs. This supports the conclusion that MMP-17 may be a member of an intracellular signaling pathway which downregulates MMP-11 mRNA.

Additional MDA‐MB‐231 breast cancer cell matrix metalloproteinases promote invasiveness

Small RNAs in the nucleus and cytoplasm of hela cells

It has been previously shown that newly synthesized nuclear low molecular weight RNA species C and D are first detected in the cytoplasm for a few minutes before they are finally found in the nucleus. The following are some of the observations made in the present study, regarding the formation of C and D RNA: (1) The 5′ end cap ribose methylation of the C RNA precursor is complete in its cytoplasmic stage; the internal ribose methylation of the precursor seems to be completed about the time of its apparent transition from cytoplasm to nucleus. (2) The few nucleotides lost from the D RNA precursor during maturation seem to be excised sometime near its apparent cytoplasmic nuclear transition. Newly synthesized C RNA also appears to lose some of its non-conserved nucleotides about the time of that transition, while the other extra nucleotides are lost later, in the nucleus. (3) The maturation of C and D RNA is inhibited early during suppression of protein synthesis by cycloheximide, while their synthesis is not. (4) The cytoplasmic precursors of C and D RNA are not associated with ribonucleoprotein particles as large as those reported for mature C and D RNA, although they do not appear to be free in the cytoplasm. (5) When the cellular UTP pool is depleted by exposure of the cells to amino sugars, and the synthesis of C, D, and other RNA species decreases, the level of[3H]uridine labeling of C and D RNA increases, while that of 4 S and 5 S RNA does not. These data are compatible with the existence of more than one nuclear UTP pool.

George L. Eliceiri

Papers

Two types of ribosome in mouse-hamster hybrid cells.

Short-lived, small RNAs in the cytoplasm of HeLa cells

Additional MDA‐MB‐231 breast cancer cell matrix metalloproteinases promote invasiveness

Small RNAs in the nucleus and cytoplasm of hela cells

Formation of low molecular weight RNA species in HeLa cells