lipid-a and Leukemia--Myelomonocytic--Acute

It has not clearly been elucidated how differently differentiation-inducing drugs act on tumor cells, whether they promote differentiation or apoptosis. To elucidate the mechanisms whether leukemic cells responding to ONO-4007, a lipid A derivative, undergo differentiation or apoptosis, we established two cell clones from a rat myelomonocytic leukemia c-WRT-7/P2 clone which undergoes differentiation followed by apoptosis by ONO-4007-treatment. One of the clones (1D6) showed the features of differentiation, such as phagocytosis when treated with ONO-4007 more than 24 hrs. The other clone (3B1) clearly showed the features of apoptosis, such as DNA ladder formation within 24 hrs after incubation with ONO-4007. We then examined expression of CD14, p21, p38MAPK, JNK/SAPK, and bcl-2, functional p53 statuses and cell cycle in these two clones, and revealed the following: Without treatment with ONO-4007; 1) CD14, p21, and bcl-2 proteins were equally expressed in both clones; 2) wild-type and non-functional mutated-type p53 were present in both clones and the p53 in 3B1 clone was recessive whereas that in 1D6 clone was dominant negative; 3) p38MAPK in 3B1 clone was already phospholyrated whereas that in 1D6 clone was not. After treatment with ONO-4007; 1) neither expressions of CD14 nor that of p21 protein was changed in any of the clones; 2) p38MAPK in 3B1 clone was dephospholyrated at 1 and 2 hrs after treatment whereas that in 1D6 clone was phospholyrated at 4 and 8 hrs after treatment; 3) the expression of bcl-2 protein in 3B1 clone was reduced. These findings suggest that p53 may be one of the key factors in leading these cells to differentiation or apoptosis, and that bcl-2 may suppress the apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Differentiation; Clone Cells; Leukemia, Myelomonocytic, Acute; Lipid A; Mutation; Proto-Oncogene Proteins c-bcl-2; Rats; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We compared the calcium mobilization in parent lipid A-sensitive leukemia cells (P2) and lipid A-resistant cells (LR) after treating them with lipid A in order to clarify the signal transduction involved in the differentiation induced by lipid A. Lipid A induced differentiation in P2 cells; however, LR cells were completely resistant to it. A dramatic elevation of intracellular free calcium ion concentration ([Ca2+]i) occurred in P2 cells, but only a slight elevation of [Ca2+]i in LR cells. Calcium ionophore in combination with lipid A induced differentiation in LR cells. An elevation of [Ca2+]i observed in P2 cells was abrogated by an addition of EGTA, which partially inhibited the differentiation of P2 cells stimulated by lipid A. Altogether, these data indicate that calcium influx is essential for the differentiation of P2 cells stimulated by lipid A and that defective calcium influx is responsible for the resistance to lipid A in LR cells.

Topics: Animals; Calcium; Cell Differentiation; Drug Resistance, Neoplasm; Egtazic Acid; Enzyme Inhibitors; Fura-2; Ionophores; Isoflavones; Leukemia, Experimental; Leukemia, Myelomonocytic, Acute; Lipid A; Naphthalenes; Rats; Rats, Inbred Strains; Receptors, Immunologic; Sulfonamides; Tumor Cells, Cultured; Vasodilator Agents

In order to demonstrate possible roles of retrodifferentiation in relapses after differentiation therapies, we have established a retrodifferentiated cell line (RD-1) from a single rat myelomonocytic leukemia cell which differentiated into a macrophage-like cell by treatment with lipopolysaccharide (LPS). The established RD-1 cells showed microscopic features slightly maturer than their parent cells. The RD-1 cells had the ability to differentiate into macrophage-like cells by treatment with fewer doses of LPS than those for parent cells. All rats inoculated with the parent cells (more than 10(2)/rat) died within 50 days. Rats inoculated with 10(4) RD-1 cells survived for more than 120 days, whereas two out of four rats inoculated with 10(5) cells and all the rats inoculated with 5 x 10(5) cells died of leukemia. These results suggest that RD-1 cells are retrodifferentiated cells from a single rat myelomonocytic leukemia cell which differentiated into a macrophage-like cell; they have similar phenotypes and lower tumorigenicity than the parent cells and they also suggest that the appearance of retrodifferentiated leukemia cells may be responsible for relapse after differentiation therapy for leukemia in some cases.

Topics: Animals; Cell Differentiation; Leukemia, Myelomonocytic, Acute; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Macrophages; Rats; Receptors, Immunologic; Tumor Cells, Cultured